Methods and compositions for regulated transcription and expression of heterologous genes

ABSTRACT

Regulatory regions from genes expressed during a particular developmental stage or in a specific tissue are identified employing cDNA screening. The resulting regulatory regions are manipulated for use with foreign sequences for introduction into plant cells to provide transformed plants having phenotypic property which can be modulated. The invention is exemplified with light, seed and fruit-specific promoters.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/105,852, filed Aug. 10, 1993, pending, which is a continuation inpart of Ser. No. 07/526,123, filed May 21, 1990, pending, which is acontinuation of Ser. No. 07/267,865, filed Nov. 7, 1988, abandoned,which is a continuation of Ser. No. 06/692,605, filed Jan. 17, 1985,abandoned. U.S. Ser. No. 08/105,852 is also a continuation in part ofSer. No. 07/582,241, filed Sep. 14, 1990, now abandoned, which is acontinuation of Ser. No. 07/188,361, filed Apr. 29, 1988, now abandoned,which is a continuation in part of Ser. No. 07/168,190, filed Mar. 15,1988, now abandoned, which is a continuation in part of Ser. No.07/054,369, filed May, 26, 1987, which issued on Jul. 24, 1990 as U.S.Pat. No. 4,943,674. U.S. Ser. No. 08/105,852 is also a continuation inpart of Ser. No. 07/742,834, filed Aug. 8, 1991, now U.S. Pat. No.5,420,034, issued on May 30, 1995, which is a continuation in part ofSer. No. 07/550,804, filed Jul. 9, 1990, now abandoned, which is acontinuation in part of Ser. No. 07/147,781, filed Jan. 25, 1988, nowabandoned, which is a continuation in part of Ser. No. 07/078,538, filedJul. 28, 1987, now abandoned, which is a continuation in part of Ser.No. 06/891,529, filed Jul. 31, 1986, now abandoned.

INTRODUCTION TECHNICAL FIELD

This invention relates to regulated genetic modification of plantmaterial, particularly for tissue and/or developmental specifictranscription and expression. Heterologous constructs are providedwhereby production of endogenous products can be modulated or newcapabilities provided.

BACKGROUND OF THE INVENTION

While the ability to manipulate bacterial and mammalian cells by hybridDNA technology has been available for almost a decade, only in 1983 wasit first reported that successful expression of an exogenous gene wasachieved in a plant cell. Plants have a highly complex genome and differin numerous ways from both bacterial and mammalian genes. Therefore,while as a first approximation, one may extrapolate from the experiencewith other species, the relevance of such experience must be determinedby experimentation. In general, genetic engineering techniques have beendirected to modifying the phenotype of individual prokaryotic andeukaryotic cells, especially in culture. Plant cells have proven moreintransigent than other eukaryotic cells due not only to the lack ofsuitable vector systems but also a result of the different goalsinvolved. Plant genetic engineering has for the most part been directedto modifying the entire plant or a particular tissue rather thanmodifying a single cell in culture.

In order to be able to successfully modify plant cells, it will benecessary to develop a large number of different systems for introducingthe exogenous DNA into the plant cell, for directing, as appropriate,the introduced DNA either randomly or to particular genomic sites, toprovide for constitutive or regulated expression and, as appropriate, toprovide for transport of the product to an appropriate site. Toward thisend, it will be necessary to develop a wide variety of regulatorysignals involved with replication, transcription, translation,integration, and the like. To varying degrees these regulatory signalswill have general application across species or be species-specific,will be associated with specific stages of plant growth, or be subjectto external control. To that extent, it will be necessary to develop awide spectrum of regulatory sequences to allow for expression underpredetermined conditions.

For many applications, it will be desirable to provide for transcriptionin a particular plant tissue and/or at a particular time in the growthcycle of the plant or maturation cycle of the tissue. Toward this end,there is substantial interest in identifying endogenous plant productstranscription or expression of which is regulated in a manner ofinterest. In identifying such products, one must first look for aproduct which appears at a particular time in the cell growth cycle orin a particular plant tissue, demonstrate its absence at other times orin other tissue, identify nucleic acid sequences associated with theproduct and then identify the sequence in the genome of the plant inorder to obtain the 5'-untranslated sequence associated withtranscription. Identifying the particular sequence, followed byestablishing that it is the correct sequence and isolating the desiredtranscriptional regulatory region requires an enormous outlay in timeand effort. One must then prepare appropriate constructs, anddemonstrate that the constructs are efficacious in the desired manner.

There has been substantial interest in modifying the seed withtranscriptional initiation regions to afford transcription andexpression of the gene introduced into the seed, rather thanconstitutive expression which would result in expression throughout theplant. Also of interest is the ability to change the phenotype of fruit,so as to provide fruit which will have improved aspects for storage,handling, cooking, organoleptic properties, freezing, nutritional value,and the like.

In addition, different systems may be required for the introduction ofnucleic acid into plant cells to obtain reasonable efficiencies oftransformation and functioning of the nucleic acid. In many instances,such as the tumor inducing plasmids and viruses, the vectors have foundlimited utilization in their range of hosts. Therefore, differenttransformation and replication systems may be required for differentplant species.

Relevant Literature

Lack of transformation by Agrobacterium of soybean is reported byDeCleene and DeLey, The Botanical Review (1976) 42:389-446. Encouragingresults in the transformation (Pederson et al., Plant Cell Repts. (1983)2:201-204 and Hood et al., Bio/Technology (1984) 2:702-708) andregeneration (Christianson et al., Science (1983) 222: 632-634) ofsoybean have recently been reported. A light inducible soybean SSU gene(small subunit SSU) of ribulose-1,5-bisphosphate-carboxylase(RuBP-carboxylase) is reported by Berry-Lowe et al., J. Mol. Appln. Gen.(1982) 1:483-498. Sequences 5' to the PSSU gene were recently shown todirect foreign gene expression in a light-inducible manner whentransferred into tobacco callus (Herrera-Estrella et al., Nature (1984)310:115-120).

Crouch et al., In: Molecular Form and Function of the Plant Genome, eds.van Vloten-Doting, Groot and Hall, Plenum Publishing Corp. 1985, pp555-566; Crouch and Sussex, Planta (1981) 153:64-74; Crouch et al., J.Mol. Appl. Genet. (1983) 2:273-283; Simon et al., Plant MolecularBiology (1985) 5:191-201; and Scofield and Crouch, J. Biol. Chem. (1987)262:12202-12208, describe various aspects of Brassica napus storageproteins. Rose et al., Nucl. Acids Res. (1987) 15:7197 and Scherer andKnauf, Plant Mol. Biol. (1987) 9:127-134 describe ACP genes. Beachy etal., EMBO J. (1985) 4:3047-3053; Sengupta-Gopalan et al., Proc. Natl.Acad. Sci. USA (1985) 82:3320-3324; Greenwood and Chrispeels, PlantPhysiol. (1985) 79:65-71 and Chen et al., Proc. Natl. Acad. Sci. USA(1986) 83:8560-8564 describe studies concerned with seed storageproteins and genetic manipulation. Eckes et al., Mol. Gen. Genet. (1986)205:1422 and Fluhr et al., Science (1986) 232:1106-1112 describe thegenetic manipulation of light inducible plant genes.

cDNA clones from tomato displaying differential expression during fruitdevelopment have been isolated and characterized (Mansson et al., Mol.Gen. Genet. (1985) 200:356-361; Slater et al., Plant Mol. Biol. (1985)5:137-147). The studies have focused primarily on mRNAS which accumulateduring fruit ripening. One of the proteins encoded by theripening-specific cDNAs has been identified as polygalacturonase (Slateret al., Plant Mol. Biol. (1985) 5:137-147). A cDNA clone which encodestomato polygalacturonase has been sequenced. Grierson et al., NucleicAcids Research (1986) 14:8395-8603. The concentration ofpolygalacturonase mRNA increases 2000-fold between the immature-greenand red-ripe stages of fruit development. This suggests that expressionof the enzyme is regulated by the specific mRNA concentration which inturn is regulated by an increase in transcription. Della Penna et al.,Proc. Natl. Acad. Sci. USA (1986) 83:6420-6424. Mature plastid mRNA forpsbA (one of the components of photosystem II) reaches its highest levellate in fruit development, whereas after the onset of ripening, plastidmRNAS for other components of photosystem I and II decline tonondetectable levels in chromoplasts. Piechulla et al., Plant Mol. Biol.(1986) 7:367-376.

SUMMARY OF THE INVENTION

Novel methods and DNA constructs are provided for transforming plantsemploying T-DNA and a Ti- or Ri-plasmid for heterologous DNAintroduction and integration into the plant genome. Transformationwithout gall formation of plant cells which have historically not beenAgrobacterium hosts is achieved with successful expression of theheterologous DNA. Additionally, DNA constructs are provided which areemployed in manipulating plant cells to provide for regulatedtranscription, such as light inducible transcription, in a plant tissueor plant part of interest at particular stages of plant growth or inresponse to external control. Particularly, transcriptional regions fromseed storage proteins, seed coat proteins or acyl carrier protein arejoined to other than the homologous gene and introduced into a plantcell host for integration into the genome to provide for seed-specifictranscription. The constructs provide for modulation of expression ofendogenous products as well as production of exogenous products in theseed. Novel DNA constructions also are provided employing afruit-specific promoter, particularly a promoter from a gene activebeginning at or shortly after anthesis or beginning at the breakerstage, joined to a DNA sequence of interest and a transcriptionaltermination region. A DNA construct may be introduced into a plant cellhost for integration into the genome and transcription regulated at atime at or subsequent to anthesis. In this manner, high levels of RNAand, as appropriate, polypeptides, may be achieved during formationand/or ripening of fruit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial sequence of the promoter region of the XBnNa napingene. The start (ATG) of the open reading frame is underlined.

FIG. 2 is a restriction map of cloned λCGN1-2 showing the entire codingregion sequence as well as extensive 5' upstream and 3' downstreamsequences.

FIG. 3 is a partial nucleotide sequence of genomic ACP clone Bcg4--4.The coding region is indicated by the three-letter amino acid codes.Breaks in the coding region sequence represent introns. The underlinednucleotide at position 310 is ambiguous without further sequenceanalysis for confirmation.

FIG. 4 is the complete nucleotide sequence of B. campestris cDNA EA9.The longest open reading frame is designated by the three letter aminoacid code. PolyA tails are evident at the end of the sequence and apotential polyadenylation signal is underlined.

FIG. 5 shows the nucleotide sequence of the cDNA clones PCGN1299 (2A11)and PCGN1298 (3H11). The amino acid sequence of the polypeptide encodedby the open reading frame is also indicated.

FIG. 6 is a comparison of 2A11 to pea storage proteins and otherabundant storage proteins:

(a) 2A11 (residues 33-46) is compared to PA1b and the reactive sitesequences of some protease inhibitors, Pa1b (residues 6-23), chick peainhibitor (residues 11-23), lima bean inhibitor (residues 23-35), humanα1-antitrypsin reactive site peptide. The arrow indicates the reactivesite.

(b) is a comparison of the amino terminal sequence of 2A11 with theamino termini of a range of seed proteins. The data have been modifiedor deletions introduced to maximize homology; conserved residues areshown boxed. The sequences are from the following sources: PA1b; barleychloroform/methanol-soluble protein d; wheat albumin; wheat α-amylaseinhibitor 0.28; millet bi-functional inhibitor; castor bean 2S smallsubunit; and napin small subunit.

FIG. 7 shows the complete sequence of the 2A11 genomic DNA cloned intoPCGN1273 from the XhoI site (position 1 at the 5' end) to the EcoRI site(position 4654).

FIG. 8 shows the nucleotide sequence of a polygalacturonase (PG) genomicclone.

FIG. 9 shows 2A11 genomic constructs. The upper line shows a map of the2A11 genomic clone. The transcriptional start site, the polyadenylationsite, the start (ATG) and stop (TGA) sites and the position of theintron are indicated. The hatched region indicates the portion of thegenomic clone that was used to make the tagged 2A11 constructions. Thebottom portion shows the regions used to construct the 2A11 cassettesincluding the synthetic oligonucleotide used to insert restriction sitesand reconstruct the 3' end.

FIG. 10 shows examples of 2A11 cassettes. Four versions of the 2A11cassette are shown. They differ only in the flanking poly-linker regionsand in the antibiotic resistance marker on the plasmid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the subject invention, DNA constructs are providedwhich allow for regulated modification of plant phenotype for exampleduring fruit development and ripening, in specific plant structuresderived from the ovum, and in chloroplast containing plant tissues suchas leaves. The DNA constructs comprise a regulated transcriptionalinitiation region. Downstream from the regulated transcriptionalinitiation region will be a sequence of interest which will provide forregulated modification of plant phenotype, by modulating the productionof an endogenous product, as to amount, relative distribution, or thelike, or production of an exogenous expression product to provide for anovel function or product. Thus genes of interest as a source ofregulated transcriptional initiation regions include those genesassociated with seed formation, preferably in association withembryogenesis and seed maturation and those associated with fruitmaturation and ripening, fruit rotting and lightinduced processes inchloroplasts. The transcriptional cassette will include in the 5'-3'direction of transcription, a regulated transcriptional andtranslational initiation region, a sequence of interest, and atranscriptional and translational termination region functional inplants. One or more introns may be also present.

In addition to the transcription construct, depending upon the manner ofintroduction of the transcription construct into the plant, other DNAsequences may be required. The subject invention includes a novel methodprovided for the introduction of foreign DNA employing T-DNA from anAgrobacterium plasmid, where efficient functional introduction ofheterologous DNA is achieved in plants normally considered outside theAgrobacterium range, e.g., monocotyledons and leguminous dicotyledons,without gall formation. The method can also be used with the knowndicotyledon hosts of Agrobacterium. DNA constructs are made which can beinserted into an Agrobacterium plasmid for transfer to a plant host.Plant hosts of particular interest are the grains and legumes.

When using the Ti- or Ri-plasmid for transformation of plant cells, asdescribed below, at least the right border and frequently both the rightand left borders of the T-DNA of the Ti- or Ri-plasmids will be joinedas flanking regions to the transcription construct. The use of T-DNA asa flanking region in a construct for integration into a Tior Ri- plasmidhas been described in EPO Application No. 116,718 and PCT ApplicationNos. W084/02913, 02919 and 02920. See also Herrera-Estrella, Nature(1983) 303:209-213; Fraley et al., Proc. Natl. Acad. Sci, USA (1983)80:4803-4807; Horsch et al., Science (1984) 223:496-498; and DeBlock etal., EMBO J. (1984) 3:1681-1689. Various fragments may be employed inthe constructions to provide for homology with the T-DNA of the tumorplasmids. The homology may involve structural genes, promoter regions,other untranslated regions such as border regions, or the like.

Downstream from and under the transcriptional initiation regulation ofthe regulatable initiation region will be a sequence of interest whichwill provide for modification of the phenotype of the specific planttissue or part. Desirably, integration constructs may be prepared whichallow for integration of the transcriptional cassette into the genome ofa plant host. Conveniently, the vector may include a multiple cloningsite downstream from the regulated transcriptional initiation region, sothat the integration construct may be employed for a variety ofsequences in an efficient manner. The DNA construct will also providefor a termination region, so as to provide an expression cassette intowhich a gene may be introduced. Conveniently, transcriptional initiationand termination regions may be provided separated in the direction oftranscription by a linker or polylinker having one or a plurality ofrestriction sites for insertion of the gene to be under thetranscriptional regulation of the regulatory regions. Usually, thelinker will have from 1 to 10, more usually from about 1 to 8,preferably from about 2 to 6 restriction sites. Generally, the linkerwill be fewer than 100 bp, frequently fewer than 60 bp and generally atleast about 5 bp. In conjunction with the subject method theseconstructs may be used for the introduction of the structural gene intoplant cells in culture, where the cells may be regenerated into wholeplants.

The DNA constructs which are provided employ T-DNA flanking regions,flanking a structural gene including transcriptional and translationalregulatory sequences. Thus, the construct which includes the structuralgene, its transcriptional and translational regulatory controls, and theT-DNA flanking regions will for the most part have the followingformula:

    (T.sup.1).sub.a -P-S.G.-Te-(T.sup.2).sub.b

wherein:

T¹ and T² are the same or different and are T-DNA from a Ti- plasmid oran Ri- plasmid, where a and b and 0 or 1, at least 1 of a and b being 1;

P is a promoter region recognized by a plant host, which promoter regionmay include promoters derived from Tior Ri- plasmids, such as theoctopine synthase or nopaline synthase promoters, viral promoters, plantpromoters, particularly leguminous and monocotyledonous plant hostpromoters of various structural genes, e.g., RuBPcarboxylase, moreparticularly SSU. The promoter region will normally include a region forbinding of RNA polymerase, as well as a cap site. In addition, there maybe present enhancers, operators, activators, or other regions involvedwith transcriptional regulation. The transcriptional initiation regionmay be native or homologous to the host or foreign or heterologous tothe host. By foreign is intended that the transcriptional initiationregion is not found in the wild-type host into which the transcriptionalinitiation region is introduced.

S.G. intends a structural gene having an open reading frame and havingat its 5'-end an initiation codon and at its 3'-end one or more nonsensecodons. The DNA sequence may have any open reading frame encoding apeptide of interest, e.g. an enzyme, or a sequence complementary to agenomic sequence, where the genomic sequence may be an open readingframe, an intron, a non-coding leader sequence, or any other sequencewhere the complementary sequence will inhibit transcription, messengerRNA processing, e.g. splicing, or translation. The DNA sequence ofinterest may be synthetic, naturally derived, or combinations thereof.Depending upon the nature of the DNA sequence of interest, it may bedesirable to synthesize the sequence with plant preferred codons. Theplant preferred codons may be determined from the codons of highestfrequency in the proteins expressed in the largest amount in theparticular plant species of interest.

Te intends a termination region functional in the plant host cell. Thetermination region, besides including at least one terminating sequence,may also include a polyA signal. The termination region which isemployed will be primarily one of convenience, since the terminationregions appear to be relatively interchangeable. The termination regionmay be native with the transcriptional initiation region, may be nativewith the DNA sequence of interest, or may be derived from anothersource. Convenient termination regions are available from the Ti-plasmidof A. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions.

Identifying useful regulated transcriptional initiation regions may beachieved in a number of ways. For example, where a fruit or seed proteinhas been or is isolated, it is partially sequenced, so that a probe canbe designed for identifying messenger RNA specific for fruit or seed. Tofurther enhance the concentration of the messenger RNA specificallyassociated with fruit or seed, cDNA can be prepared and the cDNAsubtracted with messenger RNA or cDNA from non-seed or non-fruitassociated cells. The residual CDNA can then be used for probing thegenome for complementary sequences, using an appropriate libraryprepared from plant cells. Sequences which hybridize to the cDNA thencan be isolated, manipulated, and the 5' untranslated region associatedwith the coding region isolated and used in expression constructs toidentify the transcriptional activity of the 5'-untranslated region. Insome instances, a probe may be employed directly for screening a genomiclibrary and identifying sequences which hybridize to the probe. Thesequences will be manipulated as described above to identify the5'-untranslated regions.

As an example, a promoter of particular interest for the subjectinvention, the fruit-specific transcriptional initiation region(promoter) from a DNA sequence which encodes a protein described as 2A11in the experimental section was identified as follows. cDNA clones madefrom ripe fruit were screened using cDNA probes made from ripe fruit,green fruit, and leaf mRNA. Clones were selected having more intensehybridization with the fruit DNAs as contrasted with the leaf cDNAs. Thescreening was repeated to identify a particular cDNA referred to as 2A11. The 2A11 cDNA was then used for screening RNA from root, stem, leaf,and seven stages of fruit development after the mRNA was sized on gels.The screening demonstrated that the particular message was presentthroughout the seven stages of fruit development. The MRNA complementaryto the specific cDNA was absent in other tissues which were tested. ThecDNA was then used for screening a genomic library and a fragmentselected which hybridized to the subject cDNA. The 5' and 3' non-codingregions were isolated and manipulated for insertion of a foreignsequence to be transcribed under the regulation of the 2A11 promoter.

The expression constructs which are prepared employing the regulated5'-untranslated regions may be transformed into plant cells as describedpreviously for evaluation of their ability to function with aheterologous structural gene (i.e., a gene other than the open readingframe associated with the 5'-untranslated region) and specificity ofexpression for example in a particular plant tissue or plant part suchas leaves, seed or fruit. In this manner, specific sequences may beidentified for use with sequences for fruit or seed-specifictranscription and light-induced transcription.

Several promoters are of particular interest. These include the soybeanSSU promoter, promoters from genes encoding storage proteins and seedembryo genes and those from genes that are activated at or shortly afteranthesis. The transcriptional initiation region may be native orhomologous to the host or foreign or heterologous to the host. Byforeign relative to a particular host is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced. Otherfruit-specific promoters may be activated at times subsequent toanthesis, such as prior to or during the green fruit stage, duringpre-ripe (e.g., breaker) or even into the red fruit stage.

By use of the soybean SSU promoter, it is found that the expression ofthe gene under the SSU promoter can be light-induced. Thus, theexpression of the gene is regulatable, where enhanced expression occursduring irradiation with light, while substantially reduced expression orno expression occurs in the absence of light. The nucleotide sequence ofthe small subunit gene is described by Berry-Lowe, J. Mol. Appl. Gen.(1982) 1:483-498. A DdeI digest of a plasmid containing a genomicfragment which includes the SSU soybean gene yields a 1.1 kd 5' piecethat can be used as a promoter fragment.

Transcriptional initiation regions from genes encoding storage proteins,such as those from genes encoding napin, cruciferin, β-conglycinin,phaseolin, or the like, and proteins involved in fatty acidbiosynthesis, such as acyl carrier protein (ACP) are also of interest.The transcriptional initiation regions may be obtained from anyconvenient host, particularly plant hosts such as Brassica, e.g. napusor campestris, soybean (Glycine max), bean (Phaseolus vulgaris), corn(Zea mays), cotton (Gossypium sp.), safflower (Carthamus tinctorius),tomato (Lycopersicon esculentum), and Cuphea species.

Other transcriptional initiation regions of particular interest arethose associated with seed embryo genes that are expressed in the periodfrom about day 7 to day 40, particularly those having maximum expressionin the period from about day 10 to about day 30, postanthesis, and seedcoat genes which are expressed in the period from about day 11 to day30. Usually the period of expression will be at least 3 days, moreusually about 7 days and may be substantially over the entire period.

Also of interest is a transcriptional initiation region which isactivated at or shortly after anthesis, so that in the early developmentof the fruit, it provides the desired level of transcription of thesequence of interest. Normally, the sequence of interest will beinvolved in affecting the process in the early formation of the fruit orproviding a property which is desirable during the growing (expansion)period of the fruit, or at or after harvesting.

The ripening stages of the tomato may be broken down into mature green,breaker, turning, pink, light red and red. Desirably, thetranscriptional initiation region maintains its activity during theexpansion and maturation of the green fruit, more desirably continuesactive through the ripening or red fruit period. Comparable periods forother fruit are referred to as stages of ripening. The invention is notlimited to those transcriptional initiation regions which are activatedat or shortly after anthesis but also includes transcriptionalinitiation regions which are activated at any of the ripening stages ofthe fruit. An example of a fruit-specific transcriptional initiationregion is the one referred to as 2A11 which regulates the expression ofa 2A11 cDNA sequence described in the Experimental section. The 2A11transcriptional initiation region provides for an abundant messenger,being activated at or shortly after anthesis and remaining active untilthe red fruit stage. The expressed protein is a sulfur-rich proteinsimilar to other plant storage proteins in sulfur content and size.

Also of interest is a transcriptional initiation region which regulatesexpression of the enzyme polygalacturonase, an enzyme which plays animportant role in fruit softening and/or rotting. The polygalacturonasepromoter is active in at least the breaker through red fruit stage intomato fruit.

Any structural gene of interest may be employed for use in theconstruct. In many instances, it will be desirable to have anotherstructural gene to serve as a marker associated with the construct, sothat one can detect those plant cells in which the foreign gene has beenstably introduced. For the most part, these constructs will have thefollowing formula:

    (T.sup.1).sub.a -P.sup.1 -(S.G.).sup.1 -Te.sup.1)-(.sup.2 -(S.G.).sup.2 -Te.sup.2).sub.b

wherein:

all of the symbols have the same functional definition except that thesuperscripts for P and Te intend that the promoter and terminatorregions may be the same or different, where one is a marker and theother is a structural gene of interest. Of course, one may provide for astring of expression constructs having a plurality of the same ordifferent genes in the construct. Thus, the presence of only two genesflanked by the T-DNA is merely illustrative.

As markers for structural genes, one can employ antibiotic resistancegenes, e.g., a kanamycin resistance gene or methotrexate resistance gene(DHFR). These genes are described in Haas and Dowding, supra. Othermarkers include resistance to a biocide, particularly an antibiotic,such as G418, bleomycin, hygromycin, chloramphenicol, or the like. Theparticular market employed will be one which will allow for selection oftransformed cells as compared to cells lacking the DNA which has beenintroduced.

The structural gene of interest may be any gene, either native, mutantnative, or foreign to the plant host, and may be provided in a sense orantisense orientation. For native and mutant genes, the gene may providefor increased capability of protein storage, improved nutrient source,enhanced response to light, enhanced dehydration resistance, e.g., toheat, salinity or osmotic pressure, herbicide resistance, e.g.,glyphosate, or the like. Foreign genes may include enhancement of nativecapabilities, herbicide resistance, resistance to various pests, such asviruses, insects, bacteria or fungi, production of foreign products, asa result of expression of one or more foreign genes, or the like.

In preparing the cassette construct, the various DNA sequences maynormally be inserted or substituted into a bacterial plasmid. Anyconvenient plasmid may be employed, which will be characterized byhaving a bacterial replication system, a marker which allows forselection in the bacterium and generally one or more unique,conveniently located restriction sites. These plasmids, referred to asvestors, may include such vestors as pACYC184, pACYC177, pBR322, pUC9,the particular plasmid being chosen based on the nature of the markers,the availability of convenient restriction sites, copy number, and thelike. Thus, the sequence may be inserted into the vector at anappropriate restriction site(s), the resulting plasmid used to transformthe E. coli host, the E. coli grown in an appropriate nutrient mediumand the cells harvested and lysed and the plasmid recovered. One thendefines a strategy which allows for the stepwise combination of thedifferent fragments.

As necessary, the fragments may be modified by employing syntheticadapters, adding linkers, employing in vitro mutagenesis or primerrepair to introduce specific changes in the sequence, which may allowfor the introduction of a desired restriction site, for removingsuperfluous base pairs, or the like. By appropriate strategies, onedesires to minimize the number of manipulations required as well as thedegree of selection required at each stage of manipulation. After eachmanipulation, the vector containing the manipulated DNA may be cloned,the clones containing the desired sequence isolated, and the vectorisolated and purified. As appropriate, hybridization, restrictionmapping or sequencing may be employed at each stage to ensure theintegrity and correctness of the sequence.

The cassette constructs may be introduced into the plant host cell in avariety of ways, such as an insertion into a tumor- or gall-producingplasmid, as bare DNA, as an insertion in a plant DNA virus such as A.tumefaciens or A. rhizogenes as the transforming agent, protoplastfusion, injection, electroporation, etc. For transformation withAgrobacterium, plasmids can be prepared in E. coli which plasmidscontain DNA homologous with the Ti-plasmid, particularly T-DNA. Theplasmid may or may not be capable of replication in Agrobacterium, thatis, it may or may not have a broad spectrum prokaryotic replicationsystem, e.g., RK290, depending in part upon whether the transcriptionconstruct is to be integrated into the Ti-plasmid or be retained on anindependent plasmid. By means of a helper plasmid, the transcriptionconstruct may be transferred to the A. tumefaciens and the resultingtransformed organism used for transforming plant cells.

Alternatively, to enhance integration into the plant genome, terminalrepeats of transposons may be used as borders in conjunction with atransposase. In this situation, expression of the transposase should beinducible, so that once the transcription construct is integrated intothe genome, it should be relatively stably integrated and avoid hopping.

Conveniently, explants may be cultivated with A. tumefaciens or A.rhizogenes to allow for transfer of the expression cassette to the plantcells, the plant cells dispersed in an appropriate selective medium forselection, grown to callus, shoots grown and plantlets regenerated fromthe shoots by growing in rooting medium. The Agrobacterium host willcontain a plasmid having the vir genes necessary for transfer of theT-DNA to the plant cells and may or may not have T-DNA. For injectionand electroporation, disarmed Ti-plasmids (lacking the tumor genes,particularly the T-DNA region) may be used to introduce genes into theplant cell.

In accordance with the subject invention, an efficient procedure isprovided for introduction of foreign DNA into plant cells withintegration of the DNA and without gall formation, particularly as toplants which previously have been reported to be outside the host rangeof Agrobacterium. For a list of plant genera and species which are hostsand non-hosts for Agrobacterium, see De Cleene and Le Ley, The BotanicalReview (1976) 42:389-466. Of particular interest in the subjectinvention are dicotyledon legumes, such as soybean, and monocotyledongrains, such as corn, rice, wheat, barley and oats.

Where a tumor- or gall-producing plasmid, e.g., the Ri-or Ti-plasmid, isto be used to introduce the cassette into the plant cell, a binaryplasmid, which includes an Agrobacterium functional replication system,or bacterial mating may be employed, whereby the cassette-carryingplasmid is transferred from a compatible bacterium to A. rhizogenes orA. tumefaciens and the transconjugant isolated and analyzed forintegration of the cassette into the Ri- or Ti-plasmid. This can bereadily determined by various techniques, such as Southern analysis.

The Ti- or Ri-plasmid which is employed should be capable of providingfor integration of T-DNA in the host without observable symptoms oftumor or gall formation. Thus, the plasmid which is selected may betumor-producing in a conventional host, but will not produce tumors inplants normally considered not to be hosts. An illustrative plasmid ispTiA6, a wild-type plasmid. The A. rhyizogenes or A. tumefaciensbacteria containing the cassette and the Rior Ti-plasmid may now be usedfor transformation of a plant host cell.

For transformation particularly of monocatoledenous or leguminousplants, the subject method employs in vitro grown seedlings betweengreen V-E and V-1 (Fehr and Caviness, 1977, Stages of SoybeanDevelopment. Iowa State Coop. Ext. Serv., Agric. and Home Econ. Expt.Stn. Special Report 80). Thus, young plants, the hypocotyl or next leafare employed. The Agrobacterium cells are injected into the planttissue. Generally about 1-5 μ1 of 1×10⁶ to 1×10₈ cells/ml will beinjected. Injection of Agrobacterium into cotyledons, nodes andinternodes causes a visible necrosis around the wound site. No tumorformation is observed. After about one to three weeks, the explants areexcised from the tissue surrounding the site of injection andsubcultured in a hormone lacking medium. Callus is observed to grow fromsome of the explants. Opine is present in these tissues, while none isdetected in non-transformed callus.

Transformation of seed crops such as Brassica can be by any of a varietyof methods known to those skilled in the art. See, for example, Radke etal. (1988) Theor. Appl. Genet. 75:685-694 and Radke et al. (1992) PlantCell Reports 11:499-505.

The cells which have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.,Plant Cell Reports (1986) 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having the desired phenotypic characteristicidentified. Two or more generations may be grown to ensure that thesubject phenotypic characteristic is stably maintained and inherited andthen fruits or seeds harvested to ensure the desired phenotype or otherproperty has been achieved.

As a host cell, any plant variety may be employed which provides a plantpart or tissue of interest. For example, where the plant tissue ofinterest is seed, for the most part, plants will be chosen where theseed is produced in high amounts or a seed-specific product of interestis involved. Seeds of interest include the oil seeds, such as theBrassica seeds, cotton seeds, soybean, safflower, sunflower, or thelike; grain seeds, e.g., wheat, barley, rice, clover, corn, or the like.

Where the plant part is a fruit, any of a number of fruit bearing plantsmay be employed in which the plant parts of interest are derived fromthe ovary wall. These include true berries such as tomato, grape,blueberry, cranberry, currant, and eggplant; stone fruits (drupes) suchas cherry, plum, apricot, peach, nectarine and avocado; compound fruits(droplets) such as raspberry and blackberry. In hesperidium (oranges,citrus), the expression cassette might be expected to be expressed inthe "juicy" portion of the fruit. In pepos (such as watermelon,cantaloupe, honeydew, cucumber and squash) the equivalent tissue forexpression is most likely the inner edible portions, whereas in legumes(such as peas, green beans, soybeans) the equivalent tissue is the seedpod.

By use of transcription initiation regions from regulated genes, it isfound that expression of a structural gene of interest, either sense orantisense, can be regulated in a manner similar to the regulation of thegene native to the transcription initiation region. For example, by usethe soybean SSU promoter, the expression of a gene under the control ofthis promoter is induced by light. Thus, the expression of the gene isregulatable, where enhanced expression occurs during irradiation withlight, while substantially reduced expression or no expression occurs inthe absence of light. Similarly, transcription initiation regions fromgenes expressed preferentially in seed or fruit tissues may be used tocontrol of expression of desired DNA sequences in these plant tissues.

By virtue of having a regulatable promoter in the soybean plant, one canprovide for protection against herbicides, by providing aherbicide-resistant gene to be under the regulatable control of the SSUpromoter. For example, by employing a mutated aroA gene, the enzyme5-enolpyruvyl-3-phosphoshikimate synthase which is glyphosateresistantcan be produced under light induction. Thus, the soybean plant may beprotected from glyphosate, allowing for the killing of weeds employingthe glyphosate herbicide. While glyphosate may be used by itself,particularly for pre-emergent spraying and post-emergent control ofweeds, the glyphosate may also be used with other post-emergentbroadleaf herbicides, such as Basagran (bentazan), Tackle/Blazer (aciflurofen). Normally, applications will vary from about 1.25 to 1.5lbs/acre, where the herbicides may be formulated as dry or wetformulations, by themselves or in combination with other additives, suchas sticking agents, spreading agents, stabilizers, or the like. Inertpowders may be used with dry formulations.

A transcriptional initiation region may be used for varying thephenotype of the seeds. Various changes in phenotype are of interest.These include modifying the fatty acid composition in seeds, that ischanging the ratio and/or amounts of the various fatty acids, as tolength, unsaturation, or the like. Thus, the fatty acid composition maybe varied by enhancing the fatty acids of from 10 to 14 carbon atoms ascompared to the fatty acids of from 16 to 18 carbon atoms, increasing ordecreasing fatty acids of from 20 to 24 carbon atoms, providing for anenhanced proportion of fatty acids which are saturated or unsaturated,or the like. These results can be achieved by providing for reduction ofexpression of one or more endogenous products, particularly enzymes orcofactors, by producing a transcription product which is complementaryto the transcription product of a native gene, so as to inhibit thematuration and/or expression of the transcription product, or providingfor expression of a gene, either endogenous or exogenous, associatedwith fatty acid synthesis. Expression products associated with fattyacid synthesis include acyl carrier protein, acyl-ACP thioesterase,acetyl-CoA ACP transacylase, acetyl-CoA carboxylase, ketoacyl-ACPsynthases, malonyl-CoA ACP transacylase, stearoyl-ACP desaturase, andother desaturase enzymes.

A transcriptional initiation region may be employed for varying thephenotype of the fruit. Various changes in phenotype are of interest.These changes may include up- or down-regulation of formation of aparticular saccharide, involving mono- or polysaccharides, involvingsuch enzymes as polygalacturonase, levansucrase, dextransucrase,invertase, etc.; enhance lycopene biosynthesis; cytokinin and monellinsynthesis. Other properties of interest for modification includeresponse to stress, organisms, herbicides, bruising, mechanicalagitation, etc., change in growth regulators, organoleptic properties,etc. For antisense or complementary sequence transcription, the sequencewill usually be at least 12, more usually at least 16 nt. Antisensesequences of interest include those of polygalacturonase, sucrasesynthase and invertase.

Alternatively, one may provide various products from other sourcesincluding mammals, such as blood factors, lymphokines, colonystimulating factors, interferons, plasminogen activators, enzymes, e.g.superoxide dismutase, chymosin, etc., hormones, rat mammary thioesterase2, phospholipid acyl desaturases involved in the synthesis ofeicosapentaenoic acid, and human serum albumin. The level of seedproteins, particularly mutated seed proteins, having an improved aminoacid distribution which would be better suited to the nutrient value ofthe seed can also be increased. This can be achieved, for example, byinhibition of the native seed protein by producing a complementary DNAsequence to the native coding region or non-coding region, where thecomplementary sequence does not hybridize efficiently to the mutatedsequence, or inactivates the native transcriptional capability.

A protein is provided having the sequence described in the Experimentalsection designated as 2A11 . This protein could be a storage protein andbe useful in enhancing sulfur containing amino acids (cysteine andmethionine) in the diet. It can be obtained in substantially pure formby providing for expression in prokaryotes or eukaryotes, e.g., yeast byinserting the open reading frame into an expression cassette containinga transcriptional initiation region. A variety of expression cassettesare commercially available or have been described in the literature.See, for example, U.S. Pat. Nos. 4,532,207; 4,546,082; 4,551,433; and4,559,302. The product, if intracellular, may be isolated by lysing ofthe cells and purification of the protein using electrophoresis,affinity chromatography, HPLC extraction, or the like. The product maybe isolated in substantially pure form free of other plant products,generally having at least about 95% purity, usually at least about 99%purity.

The following examples are offered by way of illustration and not bylimitation.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Cloning Vectors

Cloning vectors used include the pUC vectors, pUC8 and pUC9 (Vieira andMessing, Gene (1982) 19:259-268); pUC18 and pUC19 (Norrander et al.,Gene (1983) 26:101-106; Yanisch-Perron et al., Gene (1985) 33:103-119),and analogous vectors exchanging chloramphenicol resistance (CAM) as amarker for the ampicillin resistance of the pUC plasmids described above(pUC-CAM pUC12-Cm, pUC13-Cm! Buckley, K., Ph.D. Thesis, U.C.S.D., CA1985). The multiple cloning sites of pUC18 and pUC19 vectors wereexchanged with those of PUC-CAM to create pCGN565 and pCGN566 which areCAM resistant. Also used were pUC118 and pUC119, which are respectively,pUC18 and pUC19 with the intergenic region of M13, from an HgiAI site at5465 to the AhaIII site at 5941, inserted at the NdeI site of pUC(available from Vieira J. and Messing, J. Waksman Institute, RutgersUniversity, Rutgers, N.J.)

Materials

Terminal deoxynucleotide transferase (TDT), RNaseH, E. coli DNApolymerase, T4 kinase, and restriction enzymes were obtained fromBethesda Research Laboratories; E. coli DNA ligase was obtained from NewEngland Biolabs; reverse transcriptase was obtained from Life Sciences,Inc.; isotopes were obtained from Amersham; X-gal was obtained fromBachem, Inc., Torrance, Calif.

Bacterial strains, plasmids, and media

E. coli strains MM294(F endA1 hsdR17 supE44 thi-1) (Meselson and Yuan,Nature (1968) 217:1110-1114) and 71-18 (Alac-proAB) supE thi F' lacI^(q)Z M15 proA⁺ B⁺) (Messing et al., Proc. Natl. Acad. Sci. USA (1977)74:3642-3646) were routinely used for transformations. A. tumefaciensA348 contains the octopine Ti-plasmid pTiA6 in A114 (Garfinkel andNester, J. Bacteriol. (1980) 144:732-743). pRK2073 was maintained inHB101(F- hsd^(s) 20 (r_(B) -r_(m) -) recA13 proA2 lacY1 leuB6 rpsL20 thi1 supE44) (Boyer and Rouland-Dussiox, T. Mol. Biol. (1969) 41:459).

Plasmid pRK2073 was generated by insertion of Tn7 into the Kan^(r) geneof pRK2013. (Ditta et al., Proc. Natl. Acad. Sci. USA (1980)76:1648-1652) pSR2.1 (Berry-Lowe et al., 1982, supra.) contains a 2.1EcoRI fragment of a soybean small subunit gene (SSU) in pBR325. TheBam19 fragment of pTiA6 was maintained as a 4.6kb subclone in pBR325(pNM33C-19-1) (Thomashow et al., Cell (1980) 19:729-739). pCGN464contained the 1.5kb HindIII-SalI fragment of Tn5 cloned into the sp6transcription vector pSP65 (Melton et al., Nucl. Acids Res. (1984)12:7035-7056). The pUC7 recombinant vector containing the l.OkbBglII-SmaI fragment of Tn5(pCGN546) is designated pCGN546.

E. coli were grown on LB media (Miller, 1972, Experiments in MolecularGenetics, CSH Laboratory, Cold Spring Harbor, NY). A. tumefaciens weregrown in either minimal AB medium (Chilton et al., Proc. Natl. Acad.sci. USA (1974) 71:3672-3676) or in MG/L (50% LB:50% mannitolglutamatemedium (Roberts and Kerr, Physiol. Plant Pathol. (1974) 4:81-91.

E. coli strain pCGN1299×7118 was deposited with the American TypeCulture Collection (A.T.C.C.), 12301 Parklawn Drive, Rockville,Maryland, 20852 on May 21, 1987 and given Accession No. 67408.

EXAMPLE 1

Preparation of Transformed Soybean Plants

Soybean (glycine max cv "forrest") seeds were surface sterilized (12min,5% sodium hypochlorite, 0.1% Tween 80), washed 3 times in distilledwater and germinated aseptically (1/10 MS-Gibco, 0.6% phytagar (Gibco)medium without hormones, 25° C. red light (Grolux 40W)). Agrobacteriumcontaining strains pTiCGN327 and pTiCGN609 were grown overnight (MG/Lmedium 30° C.) were injected into hypocotyl, cotyledons, node andinternode of two to three week old seedlings. Three weeks afterinjection, tissues surrounding the injection site were excised andplaced on 0.6% phytagar MS medium deprived of hormones and containing0.5g/L carbenicillin. Hormone independent, octopine positive tissueswere then transferred to liquid MS medium and analyzed for the presenceof octopine (Otten and Schilperoot, Biochem. et Biophys. Acta 1978)527:497-500). To determine kanamycin resistance, growing calli were thenplaced in light or complete darkness. Friable calli of light grown ordark grown 327 and 609 were disaggregated by filtering through a 105μnylon mesh. Samples (0.1ml packed cell volume (p.c.v.)) of finesuspensions (1-15 cells/clump were placed in the same medium containing0 to 300mg/L kanamycin. Pigmented cells were kept in the light while thenon-pigmented cells were kept in total darkness. The effects ofkanamycin on growth were measured as packed cell volume six weeks later.

DNA Isolation

The alkali-lysis procedure of Ish-Horowitz (Maniatis et al., 21982Molecular Cloning, A Laboratory Manual, CSH Laboratory, Cold SpringHarbor, N.Y.) was used for both largescale plasmid isolation and formini-prep analysis. Total DNA from A. tumefaciens was prepared asdescribed (Currier and Nester, J. Bacteriol. (1976) 126:157-165.

DNA fragments were isolated from low melt agarose gels (Sea Plaque) runin TAE buffer (0.04M Tris-acetate, 0.002M EDTA (Maniatis, supra.)without ethidium bromide. The desired fragment was extracted from theexcised agarose band by melting at 65° C. for 30 min followed by phenolextraction and ethanol precipitation.

Cloning Procedures

Restriction enzyme digestions and ligations were performed according tomanufacturer's instructions. Klenow fill-in reactions and transformationwere as described (Maniatis, supra.) When pUC9 was being transformedinto E. coli strain 71-18, X-Gal and IPTG were added to the plates asdescribed (Miller, 1972, supra). Correct insertion and orientation ofrecombinants were verified by 2 to 3 restriction digests.

The verification of the SmaI-DdeI junction in pCGN606 was done bycloning the 1.1kb BamHi-EcoRI fragment into M13mp9 (Maniatis, supra).Sequence analysis was then performed in accordance with conventionalways.

Agrobacterium matings

The pCGN609 construct was integrated into the Tiplasmid pTiA6 in athree-way mating (Comai et al., 1983, supra). Overnight E. Coli strainscontaining cultures of pCGN609 and pRK2073, respectively, were mixedwith A. tumefaciens strain A722 and spread on AB plates containing150g/ml kanamycin and 250g/ml streptomycin. Single colonies wererestreaked twice. Correct integration was verified by Southern analysisof total Agrobacterium DNA. BamHI digested DNA was probed with anick-translated 2.5 PstI-EcoRI 3' ocs fragment from pCGN607. Southernanalysis and nick translation were performed in accordance withconventional ways. RNA preparation and Northern blot analysis RNA wasprepared from soybean callus by a modification of the guanidinethiocyanate procedure of Colbert et al. (Proc. Natl. Acad. Sci. USA(1983) 80:2248-2252) in which the extraction buffer contain 4M guanidinethiocyanate, 2% lauryl sarcosine, 1% 0-mercaptoethanol, 50mM Tris, pH7.5 20mM EDTA, 1mM aurintricarboxylic acid, 0.4% antifoam A (Sigma).PolyA⁺ RNA was purified over oligo-dT cellulose (Maniatis, supra.) andNorthern gels run as previously described (Shewmaker et al., 1984,supra). 32p RNA bacterial amioglycoside phosphotransferase mRNAcomplementary to (APH(3')II-mRNA) (Herrera-Estrella et al., EMBO J.(1983) 2:987-995; Bevan et al., Nature (1983) 304:184-187) wassynthesized from BglII cut pCGN464 using a riboprobe kit (ProMegaBiotech) according to the manufacturer's instructions. The hybridizationbuffers were as suggested by the riboprobe manufacturer's withhybridization at 55° C. and washes at 60° C.

Kanamycin activity blots

The Kanamycin activity blots (Reiss et al., Gene (1984) 30:211) wereperformed as modified for plants (Schreier et al., EMBO J. (19844(1):25-32). For each sample, 0.2g of fresh soybean callus was used.

Construction of soybean ssu-Kan^(r) chimera

A soybean SSU gene (Berry-Lowe), 1982, supra) was chosen as the sourceof the 5' -promoter region. In this gene there is a DdeI site, 9pbupstream of the AUG. A DdeI digest of pSRS2.1 (Berry-Lowe, 1982, supra.)yielded a 1.1 kd 5' fragment isolated out of a low melt agarose gel. The5' 1.1kb DdeI fragment was filled in with Klenow polymerase and ligatedinto SmaI digested pUC9 (Vieira and Messing, Gene (1982) 19:259). Aclone, pCGN606 was obtained that had the SSU promoter facing theadjacent EcoRI site of pUC9.

A cassette containing the soybean 5' region and an appropriate 3' regionwas then constructed. For this cassette, the octopine synthetase (ocs)3' region was chosen as a 2.5 kb EcoRI-PstI fragment from a Bam19subclone of pTiA6 (Thomashow, 1980, supra). Since it contained regionshomologous to T-DNA, it would facilitate transfer to the Tiplasmid ofAgrobacterium. The cassette pCGN607 was obtained in a 3-way ligationwith this fragment, the 1.1 kb BamHI-EcoRI 5' soybean SSU fragment frompCGN606, and the 2.7 kb BamHI-PstI fragment of pACYC177 (Chang andCohen, J. Bacteriol. (1978) 134:1141).

The APH(3' )-II gene employed was from Tn5, which confers resistance tokanamycin both in bacteria (Haas and Dowding, Meth. Enzymology (1975)43:611-628) and plants (Herrera-Estrella, 1983, supra). A 1.0 kbBglII-SmaI fragment containing the gene was cloned into pUC7 resultingin adjacent flanking EcoRI restriction sites. The plasmid was digestedto provide a 1.0kb EcoRI fragment and this fragment ligated into EcoRIdigested pCGN607. Clones were screened for those carrying the Kan^(r)gene of Tn5 in the correction orientation. One of the clones which hadthe correct orientation was designated pCGN609. The plasmid also carriedthe kanamycin resistance gene from pACYC177 (APH(3')-I as a bacterialmarker. These two kanamycin resistance genes (APH(3')-I and -II) do notcross-hybridize at the nucleic acid level.

Following Klenow polymerase fill-in, only 9bp which are present upstreamof the AUG in native soybean SSU are lacking in pCGN609. These 9bp arereplaced with 46bp that arise from the fusion manipulations. The rest ofthe 1.1 kb soybean SSU 5' region is the same in pCGN609 as in nativesoybean.

The integration of pCGN609 into the Ti-plasmid pTiA6 was accomplished ina three-way (Comai et al., 1983) mating with pRK2073. Correctintegration was verified by Southern analysis of the resultingAgrobacterium, designated pTiCGN609. In the integration an intactoctopine synthetase region is maintained as evidenced by the detectionof octopine. Octopine was detected by fluorescence of itsphenanthroquinone adduct following paper electrophoresis of tissueextracts (10 mg).

Transformation of soybean

Transformation of soybean was performed on in vitro grown seedlings fromthe time their cotyledons turned green up to the time of the appearanceof the second internode. In every case, the injection of Agrobacteriumcaused a clearly visible necrosis around the wound site. Occasionally,after 1 to 3 weeks, roots would appear at the inoculation site.Splitting also occurred, revealing swollen tissue, but in no case wastumor noted with the Agrobacterium strains used. Explants excised fromthe tissue surrounding the site of injection were subcultured in MSmedium deprived of hormones, 0.6% phytagar, 0.5 g/L carbenicillin.Hormone-independent callus grew from some of the explants.Hormone-independent growing tissue for the presence of octopine waspositive, while no octopine was detected in non-transformed soybeantissue. All aerial parts of the soybean seedlings, cotyledons,internodes, and nodes, were able to produce transformed tissue althoughno systematic study was done to determine which of these areas is mostsusceptible to Agrobacterium.

Analysis of polyA⁺ RNA in light and dark grown tissue

The increase in SSU protein seen in a number of light grown plants wasshown to correlate with an increase in the level of SSU polyA⁺ RNA.Northern analysis of light and dark grown 609 soybean callus wasperformed to determine if an increase in APH(3' )-II polyA⁺ RNA occurredwith growth in light. The results were determined with a ³² P-RNA probespecific for APH(3')-II transcript in the sense orientation. An RNA ofthe expected size of approximately 1.6kb was seen in both cases of lightand dark. Approximately 5-10 times as much transcript was seen in thelight grown tissue as the dark grown tissue.

Presence of protein with kanamycin phosphotransferase activity

APH(3')-II (aminoglycoside phosphotransferase) inactivates kanamycin byphosphorylation. The presence of this activity can be demonstrated by anumber of assays which measure the phosphorylation of kanamycin invitro. In the assay employed (Reiss et al., Gene (1984) 30:211) extractsare run on an acrylamide gel, reacted in situ with kanamycin and γ-³²-P-ATP and then blotted to P81 (phosphocellulose) paper. For green(light grown) and white (dark grown) 609 soybean callus, activity wasseen in the green soybean at the same mobility as that observed forpurified APH(3')-II, while no detectable activity was seen in white 609tissue or in soybean transformed with an Agrobacterium lacking theAPH(3')-II gene.

Demonstration of kanamycin resistance in the transformed tissue

Greening of the soybean callus occurred spontaneously after exposure tolight. Some of the green 609 callus selected for its friability wasdisaggregated as described previously and used to analyze its resistanceto kanamycin. It was compared to similar non-pigmented tissue grown incomplete darkness. Dark grown 609 as well as control 327 tissue died inthe presence of 50 mg/L kanamycin, while the light grown tissue couldsurvive up to 300 mg/L kanamycin although its growth was slightlyinhibited at this concentration.

EXAMPLE 2

Construction of a Napin Promoter

There are 298 nucleotides upstream of the ATG start codon of the napingene on the pgNl clone, a 3.3 kb EcoRI fragment of B. napus genomic DNAcontaining a napin gene cloned into pUC8 (available from Marti Crouch,University of Indiana). pgN1 DNA was digested with EcoRI and SstI andligated to EcoRI/SstI digested pCGN706. (pCGN706 is an XhoI/PstIfragment containing 3' and polyadenylation sequences of another napincDNA clone pN2 (Crouch et al., 1983 supra) cloned in pCGN566 at the SalIand PstI sites.) The resulting clone pCGN707 was digested with SalI andtreated with the enzyme Bal31 to remove some of the coding region of thenapin gene. The resulting resected DNA was digested with SmaI after theBal3l treatment and religated. One of the clones, pCGN713, selected bysize, was subcloned by EcoRI and BamHI digestion into both EcoRI-BamHIdigested pEMBL18 (Dente et al., Nucleic Acids Res. (1983) 11:1645-1655)and pUC118 to give E418 and E4118 respectively. The extent of Bal31digestion was confirmed by Sanger dideoxy sequencing of E418 template.The Bal3l deletion of the promoter region extended only to 57nucleotides downstream of the start codon, thus containing the 5' end ofthe napin coding sequence and about 300 bp of the 5' non-coding region.E4118 was tailored to delete all of the coding region of napin includingthe ATG start codon by in vitro mutagenesis by the method of Zoller andSmith (Nucleic Acids Res. (1982) 10:6487-6500) using an oligonucleotideprimer 5' -GATGTTTTGTATGTGGGCCCCTAGGAGATC-3' . Screening for theappropriate mutant was done by two transformations into E. coli strainJM83 (Messing J., In: Recombinant DNA Technical Bulletin, NIHPublication No. 79-99, 2 No. 2, 1979, pp 434853) and SmaI digestion ofputative transformants. The resulting napin promoter clone is pCGN778and contains 298 nucleotides from the EcoRI site of pgN1 to the Anucleotide just before the ATG start codon of napin. The promoter regionwas subcloned into a chloramphenicol resistant background by digestionwith EcoRI and BamHI and ligation to EcoRI-BamHI digested pCGN565 togive pCGN779c.

Extension of the Napin Promoter Clone

pCGN779c contains only 298 nucleotides of potential 5' -regulatorysequence. The napin promoter was extended with a 1.8 kb fragment foundupstream of the 5' -EcoRI site on the original λBnNa clone. The -3.5 kbXhoI fragment of λBnNa (available from M. Crouch), which includes thenapin region, was subcloned into SalI-digested pUC119 to give pCGN930. AHindIII site close to a 5' XhoI site was used to subclone theHindIII-EcoRI fragment of pCGN930 into HindIIIEcoRI digestedBluescript+(Vector Cloning Systems, San Diego, Calif.) to give pCGN942.An extended napin promoter was made by ligating pCGN779c digested withEcoRI and PstI and pCGN942 digested with EcoRI and PstI to make pCGN943.This promoter contains -2.1 kb of sequence upstream of the original ATGof the napin gene contained on λBnNa. A partial sequence of the promoterregion is shown in FIG. 1.

Napin Cassettes

The extended napin promoter and a napin 3' regulatory region arecombined to make a napin cassette for expressing genesseed-specifically. The napin 3' -region used is from the plasmidpCGN1924 containing the XhoI-EcoRI fragment from pgN1 (XhoI site islocated 18 nucleotides from the stop codon of the napin gene) subclonedinto EcoRi-SalI digested pCGN565. HindIII-PstI digested pCGN943 andpCGN1924 are ligated to make the napin cassette pCGN944, with uniquecloning sites SmaI, SalI, and PstI for inserting genes

Construction of CDNA Library from Spinach Leaves

Total RNA was extracted from young spinach leaves in 4M guanidinethiocyanate buffer as described by Facciotti et al. (Biotechnology(1985) 3:241-246). Total RNA was subjected to oligo(dT)-cellulose columnchromatography two times to yield poly(A)⁺ RNA as described by Maniatiset al., (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York. A cDNA library was constructed in pUC13-Cmaccording to the method of Gubler and Hoffman, (Gene (1983) 25:263-269)with slight modifications. RNasin was omitted in the synthesis of firststrand cDNA as it interfered with second strand synthesis if notcompletely removed, and dCTP was used to tail the vector DNA and dGTP totail double-stranded cDNA instead of the reverse as described in thepaper. The annealed cDNA was transformed to competent E. coli JM83(Messing (1979) supra) cells according to Hanahan (J. Mol. Biol. (1983)166:557-580) and spread onto LB agar plates (Miller (1972) Experimentsin Molecular Genetics. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) containing 50 μg/ml chloramphenicol and 0.005% X-Gal.

Identification of Spinach ACP-I cDNA

A total of approximately 8000 cDNA clones were screened by performingSouthern blots (Southern, J. Mol. Biol. (1975) 98:503) and dot blot(described below) hybridizations with clone analysis DNA from 40 poolsrepresenting 200 cDNA clones each (see below). A 5' endlabeled syntheticoligonucleotide (ACPP4) that is at least 66% homologous with a 16 aminoacid region of spinach ACP-I (5'-GATGTCTTGAGCCTTGTCCTCATCCACATTGATACCAAACTCCTCCTC-3' ) is the complementto a DNA sequence that could encode the 16 amino acid peptideglu-glu-glu-phe-gly-ile-asn-val-asp-gluasp-lys-ala-gln-asp-ile, residues49-64 of spinach ACP-I (Kuo and Ohlrogge, Arch. Biochem. Biophys. (1984)234:290-296) and eas used for an ACP probe.

Clone analysis DNA for Southern and dot blot hybridizations was preparedas follows. Transformants were transferred from agar plates to LBcontaining 50 βg/ml chloramphenicol in groups of ten clones per 10 mlmedia. Cultures were incubated overnight in a 37° C. shaking incubatorand then diluted with an equal volume of media and allowed to grow for 5more hours. Pools of 200 cDNA clones each were obtained by mixingcontents of 20 samples. DNA was extracted from these cells as describedby Birnboim and Doly (Nucleic Acids Res. (1979) 7:1513-1523). DNA waspurified to enable digestion with restriction enzymes by extractionswith phenol and chloroform followed by ethanol precipitation. DNA wasresuspended in sterile, distilled water and 1 fg of each of the 40pooled DNA samples was digested with EcoRI and HindIII andelectrophoresed through 0.7% agarose gels. DNA was transferred tonitrocellulose filters following the blot hybridization technique ofSouthern.

ACPP4 was 5' end-labeled using γ³² p DATP and T4 kinase according to themanufacturer's specifications. Nitrocellulose filters from Southern blottransfer of clone analysis DNA were hybridized (24 hours, 42° C.) andwashed according to Berent et al. (BioTechniques (1985) 3:208-220). Dotblots of the same set of DNA pools were prepared by applying 1 μg ofeach DNA pool to nylon membrane filters in 0.5 M NaOH. These blots werehybridized with the probe for 24 hours at 42° C. in 50% formamide/1%SDS/1 M NaCl, and washed at room temperature in 2X SSC/0.1% SDS (1XSSC=0.15 M NaCl; 0.015 M Na citrate; SDS-sodium dodecylsulfate). DNAfrom the pool which was hybridized by the ACPP4 oligoprobe wastransformed to JM83 cells and plated as above to yield individualtransformants. Dot blots of these individual cDNA clones were preparedby applying DNA to nitrocellulose filters which were hybridized with theACPP4 oligonucleotide probe and analyzed using the same conditions asfor the Southern blots of pooled DNA samples.

Nucleotide Sequence Analysis

The positive clone, pCGNlSOL, was analyzed by digestion with restrictionenzymes and the following partial map was obtained.

    ______________________________________                                         ##STR1##                                                                      *former PstI site destroyed with tailing                                      **polylinker with available restriction sites indicated                  

The cDNA clone was subcloned into pUC118 and pUC119 using standardlaboratory techniques of restriction, ligation, transformation, andanalysis (Maniatis et al., (1982) supra). Single-stranded DNA templatewas prepared and DNA sequence was determined using the Sanger dideoxytechnique (Sanger et al., Proc. Nat. Acad. Sci. USA (1977)74:5463-5467). Sequence analysis was performed using a software packagefrom IntelliGenetics, Inc. pCGNlSOL contains an (approximately) 700 bpcDNA insert including a stretch of A residues at the 3' terminus whichrepresents the poly(A) tail of the mRNA. An ATG codon at position 61 ispresumed to encode the MET translation initiation codon. This codon isthe start of a 411 nucleotide open reading frame, of which, nucleotides229-471 encode a protein whose amino acid sequence corresponds almostperfectly with the published amino acid sequence of ACP-I of Kuo andOhlrogge supra as described previously. In addition to mature protein,the pCGNiSOL also encodes a 56 residue transit peptide sequence, asmight be expected for a nuclear-encoded chloroplast protein.

Napin-ACP Construct

pCGN796 was constructed by ligating pCGNlSOL digested withHindIII-BamHI, pUC8-CM digested with HindIII and BamHI and pUC118digested with BamHI. The ACP gene from pCGN796 was transferred into achloramphenicol background by digestion with BamHI and ligation withBamHI digested pCGN565. The resulting pCGN1902 was digested with EcoRIand SmaI and ligated to EcoRI-SmaI digested pUC118 to give pCGN1920. TheACP gene in pCGN1920 was digested at the NcoI site, filled in bytreatment with the Klenow fragment, digested with SmaI and religated toform pCGN1919. This eliminated the 5' -coding sequences from the ACPgene and regenerated the ATG. This ACP gene was flanked with PstI sitesby digesting pCGN1919 with EcoRI, filling in the site with the Klenowfragment and ligating a PstI linker. This clone is called pCGN945.

The ACP gene of pCGN945 was moved as BamHI-PstI fragment to pUC118digested with BamHI and PstI to create pCGN945a so that a SmaI site(provided by the pUC118) would be at the 5' -end of the ACP sequences tofacilitate cloning into the napin cassette pCGN944. pCGN945a digestedwith SmaI and PstI was ligated to pCGN944 digested with SmaI and PstI toproduce the napin ACP cassette pCGN946. The napin ACP cassette was thentransferred into the binary vector pCGN783 by cloning from the HindIIIsite to produce pCGN948.

Construction of the Binary Vector DCGN783

pCGN783 is a binary plasmid containing the left and right T-DNA bordersof A. tumefaciens (Barker et al., Plant Mol. Biol. (1983) 2:335-350);the gentamicin resistance gene of pPH1JI (Hirsch et al., Plasmid (1984),12:139-141) the 35S promoter of cauliflower mosaic virus (CaMV) (Gardneret al., Nucleic Acids Res. (1981) 9:2871-2890), the kanamycin resistancegene of Tn5 (Jorgenson et al., infra and Wolff et al., Nucleic AcidsRes. (1985) 13:355-367) and the 3' region from transcript 7 k of pTiA6(Barker et al., (1983) supra).

To obtain the gentamicin resistance marker, the gentamicin resistancegene was isolated as a 3.1 kb EcoRIPstI fragment of pPHIJl cloned intopUC9 yielding pCGN549. The HindIII-BamHI fragment containing thegentamicin resistance gene was substituted for the HindIII-BglIIfragment of pCGN587 creating pCGN594.

pCGN587 was prepared as follows: The HindIII-SmaI fragment of Tn5containing the entire structural gene for APHII (Jorgenson et al., Mol.Gen. Genet. (1979) 177:65) was cloned into pUC8 (Vieira and Messing,Gene (1982) 19:259), converting the fragment into a HindIII-EcoRIfragment, since there is an EcoRI site immediately adjacent to the SmaIsite. The PstI-EcoRI fragment containing the 3' -portion of the APHIIgene was then combined with an EcoRI-BamHI-SalIPstI linker into theEcoRI site of pUC7 (pCGN546W). Since this construct does not conferkanamycin resistance, kanamycin resistance was obtained by inserting theBg1IIPstI fragment of the APHII gene into the BamHI-PstI site(pCGN546X). This procedure reassembles the APHII gene, so that EcoRIsites flank the gene. An ATG codon was upstream from and out of readingframe with the ATG initiation codon of APHII. The undesired ATG wasavoided by inserting a Sau3A-PstI fragment from the 5' -end of APHII,which fragment lacks the superfluous ATG, into the BamHI-PstI site ofpCGN546W to provide plasmid pCGN550.

The EcoRI fragment containing the APHII gene was then cloned into theunique EcoRI site of pCGN451, which contains an octopine synthasecassette for expression, to provide pCGN552 (1ATG).

pCGN451 includes an octopine cassette which contains about 1556 bp ofthe 5' non-coding region fused via an EcoRI linker to the 3' non-codingregion of the octopine synthase gene of pTiA6. The pTi coordinates are11,207 to 12,823 for the 3' region and 13,643 to 15,208 for the 5'region as defined by Barker et al., Plant Mol. Biol. (1983) 2:325.

The 5' fragment was obtained as follows. A small subcloned fragmentcontaining the 5' end of the coding region, as a BamHI-EcoRI fragmentwas cloned in pBR322 as plasmid pCGN407. The BamHI-EcoRI fragment has anXmnI site in the coding region, while pBR322 has two XmnI sites. pCGN407was digested with XmnI, resected with Bal31 nuclease and EcoRI linkersadded to the fragments. After EcoRI and BamHI digestion, the fragmentswere size fractionated, the fractions cloned and sequenced. In one case,the entire coding region and 10 bp of the 5' non-translated sequenceshad been removed leaving the 5' non-translated region, the mRNA cap siteand 16 bp of the 5' non-translated region (to a BamHI site) intact. Thissmall fragment was obtained by size fractionation on a 7% acrylamide geland fragments approximately 130 bp long eluted.

This size fractionated DNA was ligated into M13mp9 and several clonessequenced and the sequence compared to the known sequence of theoctopine synthase gene. The M13 construct was designated p14, whichplasmid was digested with BamHI and EcoRI to provide the small fragmentwhich was ligated to a XhoI to BamHI fragment containing upstream 5'sequences from pTiA6 (Garfinkel and Nester, J. Bacteriol. (1980)144:732) and to an EcoRI to XhoI fragment containing the 3' sequences.

The resulting XhoI fragment was cloned into the XhoI site of a pUC8derivative, designated pCGN426. This plasmid differs from pUC8 by havingthe sole EcoRI site filled in with DNA polymerase I, and having lost thePstI and HindIII site by nuclease contamination of HincII restrictionendonuclease, when a XhoI linker was inserted into the unique HincIIsite of pUC8. The resulting plasmid pCGN451 has a single EcoRI site forthe insertion of protein coding sequences between the 5' non-codingregion (which contains 1,550 bp of 5' non-transcribed sequence includingthe right border of the T-DNA, the mRNA cap site and 16 bp of 5'nontranslated sequence) and the 3' region (which contains 267 bp of thecoding region, the stop codon, 196 bp of 3' nontranslated DNA, the polyAsite and 1,153 bp of 3' nontranscribed sequence). pCGN451 also providesthe right TDNA border.

The resulting plasmid pCGN451 having the ocs 5' and the ocs 3' in theproper orientation was digested with EcoRI and the EcoRI fragment frompCGN551 containing the intact kanamycin resistance gene inserted intothe EcoRI site to provide pCGN552 having the kanamycin resistance genein the proper orientation.

This ocs/KAN gene was used to provide a selectable marker for the transtype binary vector pCGN587.

The 5' portion of the engineered octopine synthase promoter cassetteconsists of pTiA6 DNA from the XhoI at bp 15208-13644 (Barker' snumbering), which also contains the TDNA boundary sequence (border)implicated in T-DNA transfer. In the plasmid pCGN587, the ocs/KAN genefrom pCGN552 provides a selectable marker as well as the right border.The left boundary region was first cloned in M13mp9 as a HindIII-SmaIpiece (pCGN502) (base pairs 602-2213) and recloned as a KpnI-EcoRIfragment in pCGN565 to provide pCGN580. pCGN565 is a cloning vectorbased on pUC8-Cm, but containing pUC18 linkers. pCGN580 was linearizedwith BamHI and used to replace the smaller BglII fragment of pVCK102(Knauf and Nester, Plasmid (1982) 8:45), creating pCGN585. By replacingthe smaller SalI fragment of pCGN585 with the XhoI fragment from pCGN552containing the ocs/KAN gene, pCGN587 was obtained.

The pCGN594 HindIII-BamHI region, which contains an 5'-ocs-kanamycin-ocs-3' (ocs is octopine synthase with 5' designating thepromoter region and 3' the terminator region, see U.S. applicationserial no. 775,923, filed Sep. 13, 1985) fragment was replaced with theHindIIIBamHI polylinker region from pUC18 . pCGN566 contains theEcoRI-HindIII linker of pUC18 inserted into the EcoRI-HindIII sites ofpUC13-Cm. The HindIII-Bg1II fragment of pNW31C-8,29-1 (Thomashow et al.,Cell (1980) 19:729) containing ORF1 and -2 of pTiA6 was subcloned intothe HindIII-BamHI sites of pCGN566 producing pCGN703.

The Sau3A fragment of pCGN703 containing the 3' region of transcript 7(corresponding to bases 2396-2920 of pTiA6 (Barker et al., (1983) supra)was subcloned into the BamHI site of pUC18 producing pCGN709. TheEcoRI-SmaI polylinker region of pCGN709 was substituted with theEcoRISmaI fragment of pCGN587, which contains the kanamycin resistancegene (APH3-II) producing pCGN726.

The EcoRI-SalI fragment of pCGN726 plus the BglIIEcoRI fragment ofpCGN734 were inserted into the BamHI-SalI site of pUC8-Cm producingpCGN738. pCGN726c is derived from pCGN738 by deleting the 900 bpEcoRI-EcoRI fragment.

To construct pCGN167, the AluI fragment of CaMV (bp 7144-7735) (Gardneret al., Nucl. Acid Res. (1981) 9:2871-2888) was obtained by digestionwith AluI and cloned into the HincII site of M13mp7 (Messing et al.,Nucl. Acids Res. (1981) 9:309-321) to create C614. An EcoRI digest ofC614 produced the EcoRI fragment from C614 containing the 35S promoterwhich was cloned into the EcoRI site-of pUC8 (Vieira and Messing, Gene(1982) 19:259) to produce pCGN146.

To trim the promoter region, the BglII site (bp 7670) was treated withBglII and resected with Bal31 and subsequently a BglII linker wasattached to the Bal31 treated DNA to produce pCGN147. pCGN148acontaining a promoter region, selectable marker (KAN with 2 ATG' s) and3' region, was prepared by digesting pCGN528 with BglII and insertingthe BamHI-BglII promoter fragment from pCGN147. This fragment was clonedinto the BglII site of pCGN528 so that the BglII site was proximal tothe kanamycin gene of pCGN528.

The shuttle vector used for this construct, pCGN528, was made asfollows. pCGN525 was made by digesting a plasmid containing Tn5 whichharbors a kanamycin gene (Jorgenson et al., Mol. Gen. Genet. (1979)177:65) with HindIII-BamHI and inserting the HindIII-BamHI fragmentcontaining the kanamycin gene into the HindIII-BamHI sites in thetetracycline gene of pACYC184 (Chang and Cohen, J. Bacteriol. (1978)134:1141-1156). pCGN526 was made by inserting the BamHI fragment 19 ofpTiA6 (Thomashow et al., Cell (1980) 19:729-739), modified with XhoIlinkers inserted into the SmaI site, into the BamHI site of pCGN525.pCGN528 was obtained by deleting the small XhoI fragment from pCGN526 bydigesting with XhoI and religating. pCGN149a was made by cloning theBamHI-kanamycin gene fragment from pMB9KanXXI into the BamHI site ofpCGN148a. pMB9KanXXI is a pUC4K variant (Vieira and Messing, Gene (1982)19:259-268) which has the XhoI site missing but contains a functionalkanamycin gene from Tn903 to allow for efficient selection inAgrobacterium. pCGN149a was digested with BglII and SphI. This smallBglII-SphI fragment of pCGN149a was replaced with the BamHI-SphIfragment from MI (see below) isolated by digestion with BamHI and SphI.This produces pCGN167, a construct containing a full length CaMVpromoter, 1ATG-kanamycin gene, 3' end and the bacterial Tn903-typekanamycin gene. MI is an EcoRI fragment from pCGN546X (see constructionof pCGN587) and was cloned into the EcoRI cloning site of M13mp9 in sucha way that the PstI site in the 1ATG-kanamycin gene was proximal to thepolylinker region of M13mp9.

The HindIII-BamHI fragment in the pCGN167 containing the CaMV-35Spromoter, 1ATG-kanamycin gene and the BamHIfragment 19 of pTiA6 wascloned into the BamHI-HindIII sites of pUC19 creating pCGN976. The 35Spromoter and 3' region from transcript 7 was developed by inserting a0.7 kb HindIII-EcoRI fragment of pCGN976 (35S promoter) and the 0.5 kbEcoRI-SalI fragment of pCGN709 (transcript 7:3' ) into the HindIII-SalIsites of pCGN566 creating pCGN766c.

The 0.7 kb HindIII-EcoRI fragment of pCGN766c (CaMV35S promoter) wasligated to the 1.5 kb EcoRI-SalI fragment in pCGN726c (LATG-KAN 3'region) followed by insertion into the HindIII-SalI sites of pUC119 toproduce pCGN778. The 2.2 kb region of pCGN778, HindIII-SalI fragmentcontaining the CaMV-35S promoter and 1ATG-KAN-3' region was used toreplace the HindIII-SalI linker region of pCGN739 to produce pCGN783.

Transfer of the Binary Vector pCGN948 into Agrobacterium

pCGN948 was introduced into Agrobacterium tumefaciens EHA101 (Hood etal., J. Bacteriol. (1986) 168:1291-1301) by transformation. An overnight2 ml culture of EHA101 was grown in MG/L broth at 30° C. 0.5 ml wasinoculated into 100 ml of MG/L broth (Garfinkel and Nester, J.Bacteriol. (1980) 144:732-743) and grown in a shaking incubator for 5 hat 30° C. The cells were pelleted by centrifugation at 7K, resuspendedin 1 ml of MG/L broth and placed on ice. Approximately, 1 Ag of pCGN948DNA was placed in 100 gl of MG/L broth to which 200 gl of the EHA101suspension was added; the tube containing the DNA-cell mix wasimmediately placed into a dry ice/ethanol bath for 5 minutes. The tubewas quick thawed by 5 minutes in 37° C. water bath followed by 2 h ofshaking at 30° C. after adding 1 ml of fresh MG/L medium. The cells werepelleted and spread onto MG/L plates (1.5% agar) containing 100 mg/lgentamicin. Plasmid DNA was isolated from individualgentamicin-resistant colonies, transformed back into E. coli, andcharacterized by restriction enzyme analysis to verify that thegentamicin-resistant EHA101 contained intact copies of pCGN948. Singlecolonies are picked and purified by two more streakings on MG/L platescontaining 100 mg/l gentamicin.

Transformation and Regeneration of B. Napus

Seeds of Brassica napus cv Westar were soaked in 95% ethanol for 4minutes. They were sterilized in 1% solution of sodium hypochlorite with50 Al of "Tween 20" surfactant per 100 ml sterile solution. Aftersoaking for 45 minutes, seeds were rinsed 4 times with sterile distilledwater. They were planted in sterile plastic boxes 7 cm wide, 7 cm long,and 10 cm high (Magenta) containing 50 ml of 1/10 th concentration of MS(Murashige minimal organics medium, Gibco) with added pyridoxine (50Ag/1), nicotinic acid (50 μg/l), glycine (200 Ag/l) and solidified with0.6% agar. The seeds germinated and were grown at 22° C. in a 16h-8hlight-dark cycle with the light intensity approximately 65 μEm⁻² s⁻¹.After 5 days the seedlings were taken under sterile conditions and thehypocotyls excised and cut into pieces of about 4 mm in length. Thehypocotyl segments were placed on a feeder plate or without the feederlayer on top of a filter paper on the solidified B5 0/1/1 or B5 0/1/0medium. B5 0/1/0 medium contains B5 salts and vitamins (Gamborg, Millerand Ojima, Experimental Cell Res. (1968) 50:151-158), 3% sucrose,2,4-dichlorophenoxyacetic acid (1.0 mg/l), pH adjusted to 5.8, and themedium is solidified with 0.6% Phytagar; B5 0/1/1 is the same with theaddition of 1.0 mg/l kinetin. Feeder plates were prepared 24 hours inadvance by pipetting 1.0 ml of a stationary phase tobacco suspensionculture (maintained as described in Fillatti et al., Molecular GeneralGenetics (1987) 206:192-199) onto B5 0/1/0 or B5 0/1/1 medium. Hypocotylsegments were cut and placed on feeder plates 24 hours prior toAgrobacterium treatment.

Agrobacterium tumefaciens (strain EHA101×948) was prepared by incubatinga single colony of Agrobacterium in MG/L broth at 30° C. Bacteria wereharvested 16 hours later and dilutions of 10⁸ bacteria per ml wereprepared in MG/L broth. Hypocotyl segments were inoculated with bacteriaby placing the segments in an Agrobacterium suspension and allowing themto set for 30-60 minutes, then removing and transferring to Petri platescontaining B5 0/1/1 or 0/1/0 medium (0/1/1 intends 1 mg/l 2,4-D and 1mg/l kinetin and 0/1/0 intends no kinetin). The plates were incubated inlow light at 22° C. The co-incubation of bacteria with the hypocotylsegments took place for 24-48 hours. The hypocotyl segments were removedand placed on B5 0/1/1 or 0/1/0 containing 500 mg/l carbenicillin(kanamycin sulfate at 10, 25, or 50 mg/l was sometimes added at thistime) for 7 days in continuous light (approximately 65 μEM⁻² S⁻¹) at 22°C. The segments were transferred to B5 salts medium containing 1%sucrose, 3 mg/l benzylamino purine (BAP) and 1 mg/l zeatin. This wassupplemented with 500 mg/l carbenicillin, 10, 25, or 50 mg/1 kanamycinsulfate, and solidified with 0.6% Phytagar (Gibco). Thereafter, explantswere transferred to fresh medium every two weeks.

After one month green shoots developed from green calli which wereselected on media containing kanamycin. Shoots continued to develop forthree months. The shoots were cut from the calli when they were at least1 cm high and placed on B5 medium with 1% sucrose, no added growthsubstances, 300 mg/l carbenicillin, and solidified with 0.6% phytagar.The shoots continued to grow and several leaves were removed to test forneomycin phosphotransferase II (NPTII) activity. Shoots which werepositive for NPTII activity were placed in Magenta boxes containing B50/1/1 medium with 1% sucrose, 2 mg/l indolebutyric acid, 200 mg/lcarbenicillin, and solidified with 0.6% Phytagar. After a few weeks theshoots developed roots and were transferred to soil. The plant weregrown in a growth chamber at 22° C. in a 16-8 hours light-dark cyclewith light intensity 220 EK² S-¹ and after several weeks weretransferred to the greenhouse.

Southern Data

Regenerated B. napus plants from cocultivations of Agrobacteriumtumefaciens EHA101 containing pCGN948 and B. napus hypocotyls wereexamined for proper integration and embryo-specific expression of thespinach leaf ACP gene. Southern analysis was performed using DNAisolated from leaves of regenerated plants by the method of Dellaportaet al. (Plant Mol. Biol. Rep. (1983) 1:19-21) and purified once bybanding in CsCl. DNA (10 μg) was digested with the restriction enzymeEcoRI, electrophoresed on a 0.7% agarose gel and blotted tonitrocellulose (see Maniatis et al., (1982) supra.). Blots were probedwith pCGN945 DNA containing 1.8 kb of the spinach ACP sequence or withthe EcoRI-HindIII fragment isolated from pCGN936c (made by transferringthe HindIII-EcoRI fragment of pCGN930 into pCGN566) containing the napin5' sequences labeled with ³² p-dCTP by nick translation (described bythe manufacturer, BRL Nick Translation Reagent Kit, Bethesda ResearchLaboratories, Gaithersburg, Md.). Blots were prehybridized andhybridized in 50% formamide, lox Denhardt's, 5xSSC, 0.1% SDS, 5 mM EDTA,100 μg/ml calf thymus DNA and 10% dextran sulfate (hybridization only)at 42° C. (Reagents described in Maniatis et al., (1982) supra.) Washeswere in 1xSSC, 0.1% SDS, 30 min and twice in 0.1xSSC, 0.1% SDS 15 mineach at 55° C.

Autoradiograms showed two bands of approximately 3.3 and 3.2 kbhybridized in the EcoRI digests of DNA from four plants when probed withthe ACP gene (pCGN945) indicating proper integration of the spinach leafACP construct in the plant genome since 3.3 and 3.2 kb EcoRI fragmentsare present in the T-DNA region of pCGN948. The gene construct waspresent in single or multiple loci in the different plants as judged bythe number of plant DNA-construct DNA border fragments detected whenprobed with the napin 5' sequences.

Northern Data

Expression of the integrated spinach leaf ACP gene from the napinpromoter was detected by Northern analysis in seeds but not leaves ofone of the transformed plants shown to contain the construct DNA.Developing seeds were collected from the transformed plant 21 dayspostanthesis. Embryos were dissected from the seeds and frozen in liquidnitrogen. Total RNA was isolated from the seed embryos and from leavesof the transformed plant by the method of Crouch et al., (1983) supra,electrophoresed on formaldehyde-containing 1.5% agarose gels asdescribed (Shewmaker et al., Virology (1985) 140:281-288) and blotted tonitrocellulose (Thomas, Proc. Natl. Acad. Sci. USA (1980) 77:5201-5205).Blots were prehybridized, hybridized, and washed as described above. Theprobe was an isolated PstI-BamHI fragment from pCGN945 containing onlyspinach leaf ACP sequences labeled by nick translation.

An RNA band of ˜0.8 kb was detected in embryos but not leaves of thetransformed plant indicating seed-specific expression of the spinachleaf ACP gene.

EXAMPLE 3

Construction of B. Campestris Napin Promoter Cassette

A BglII partial genomic library of B. campestris DNA was made in thelambda vector Charon 35 using established protocols (Maniatis et al.,(1982) supra). The titer of the amplified library was -1.2×10⁹ phage/ml.Four hundred thousand recombinant bacteriophage were plated at a densityof 10⁵ pre 9×9 in. NZY plate (NZYM as described in Maniatis et al.,(1982) supra) in NZY+10 mM MgSO₄ +0.9% agarose after adsorption to DHlE. coli cells (Hanahan, Mol. Biol. (1983) 166:557) for 20 min at 37° C.Plates were incubated at 37° C. for -13 hours, cooled at 4° C. for 2.5hours and the phage were lifted onto Gene Screen Plus (New EnglandNuclear) by laying precut filters over the plates for approximately 1min and peeling them off. The adsorbed phage DNA was immobilized byfloating the filter on 1.5 M NaCl, 0.5 M NaOH for 1 min., neutralizingin 1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0 for 2 min and 2XSSC for 3 min.Filters were air dried until just damp, prehybridized and hybridized at42° C. as described for Southern analysis. Filters were probed fornapin-containing clones using an XhoI-SalI fragment of the CDNA cloneBE5 which was isolated from the B. campestris seed cDNA librarydescribed using the probe pN1 (Crouch et al., (1983) supra). Threeplaques were hybridized strongly on duplicate filters and were plaquepurified as described (Maniatis et al., (1982) supra). one of the clonesnamed lambda CGN1-2 was restriction mapped and the napin gene waslocalized to overlapping 2.7 kb XhoI and 2.1 kb SalI restrictionfragments. The two fragments were subcloned from lambda CGN1-2 DNA intopCGN789 (a pUC based vector the same as pUC119 with the normalpolylinker replaced by the synthetic linker-5'GGAATTCGTCGACAGATCTCTGCAGCTCGAGGGATCCAAGCTT 3' (which represents thepolylinker EcoRI, SalI, BgiII, PstI, XhoI, BamHI, HindIII). The identityof the subclones as napin was confirmed by sequencing. The entire codingregion sequence as well as extensive 5' upstream and 3' downstreamsequences were determined (FIG. 2). The lambda CGN1-2 napin gene is thatencoding the MRNA corresponding to the BE5 cDNA as determined by theexact match of their nucleotide sequence.

An expression cassette was constructed from the 5' end and the 3' -endof the lambda CGN1-2 napin gene as follows in an analogous manner to theconstruction of pCGN944. The majority of the napin coding region ofpCGN940 was deleted by digestion with SalI and religation to formpCGN1800. Single-stranded DNA from pCGN1800 was used in an in vitromutagenesis reaction (Adelman et al., DNA (1983) 2:183-193) using thesynthetic oligonucleotide 5' GCTTGTTCGCCATGGATATCTTCTGTATGTTC 3' . Thisoligonucleotide inserted an EcoRV and an Ncol restriction site at thejunction of the promoter region and the ATG start codon of the napingene. An appropriate mutant was identified by hybridization to theoligonucleotide used for the mutagenesis and sequence analysis and namedpCGN1801.

A 1.7 kb promoter fragment was subcloned from pCGN1801 by partialdigestion with EcoRV and ligation to pCGN786 (a pCGN566 chloramphenicolbased vector with the synthetic linker described above in place of thenormal polylinker) cut with EcoRI and blunted by filling in with DNAPolymerase I Klenow fragment to create pCGN1802. 3' sequences from thelambda CGN1-2 napin gene were added to XhoI-HindIII digested pCGN1802from pCGN941 digested with XhoI and HindIII. The resulting clone,pCGN1803, contains approximately 1.6 kb of napin 3' -sequences as wellas promoter sequences, but a 326 nucleotide HindIII fragment normallyfound at the 3' -end of lambda CGN1-2 is inserted opposite to itsnatural orientation. As a result, there are two HindIII sites inpCGN1803. This reversed fragment was removed by digestion of pCGN1803with HindIII. Following religation, a clone was selected which nowcontained only approximately 1.25 kb of the original 1.6 napin 3'-sequence. This clone, pCGN1808, is the lambda CGN1-2 expressioncassette and contains 1.725 kb of napin promoter sequence, and 1.265 kbof napin 3' sequences with the unique cloning sites SalI, BglI, PstI,and XhoI in between. Any sequence that requires seed-specifictranscription or expression in Brassica, for example, a fatty acid gene,can be inserted in this cassette in a manner analogous to that describedfor spinach leaf ACP and the B. napus napin cassette (see Example 2).

pCGN3223 Napin Expression Cassette

pCGN1808 is modified to contain flanking restriction sites to allowmovement of only the expression sequences and not the antibioticresistance marker to binary vectors such as pCGN1557 (McBride andSummerfelt (1990) Pl. Mol. Biol. 14:269-276). Synthetic oligonucleotidescontaining KpnI, NotI and HindIII restriction sites are annealed andligated at the unique HindIII site of pCGN1808, such that only oneHindIII site is recovered. The resulting plasmid, pCGN3200 containsunique HindIII, NotI and KpnI restriction sites at the 3' -end of thenapin 3' -regulatory sequences as confirmed by sequence analysis.

The majority of the napin expression cassette is subcloned from pCGN3200by digestion with HindIII and SacI and ligation to HindIII and SacIdigested pIC19R (Marsh, et al. (1984) Gene 32:481-485) to make pCGN3212.The extreme 5' -sequences of the napin promoter region are reconstructedby PCR using pCGN3200 as a template and two primers flanking the SacIsite and the junction of the napin 5' -promoter and the pUC backbone ofpCGN3200 from the pCGN1808 construct. The forward primer contains ClaI,HindIII, NotI, and KpnI restriction sites as well as nucleotides 408-423of the napin 5' -sequence (from the EcoRV site) and the reverse primercontains the complement to napin sequences 718-739 which include theunique SacI site in the 5' -promoter. The PCR was performed using in aPerkin Elmer/Cetus thermocycler according to manufacturer'sspecifications. The PCR fragment is subcloned as a blunt-ended fragmentinto pUC8 (Vieira and Messing (1982) Gene 19:259-268) digested withHincII to give pCGN3217. Sequence of pCGN3217 across the napin insertverifies that no improper nucleotides were introduced by PCR. The napin5-sequences in pCGN3217 are ligated to the remainder of the napinexpression cassette by digestion with ClaI and SacI and ligation topCGN3212 digested with ClaI and SacI. The resulting expression cassettepCGN3221, is digested with HindIII and the napin expression sequencesare gel purified away and ligated to pIC20H (Marsh, supra) digested withHindIII. The final expression cassette is pCGN3223, which contains in anampicillin resistant background, essentially identical 1.725 napin 5'and 1.265 3' regulatory sequences as found in pCGN1808. The regulatoryregions are flanked with HindIII, NotI and KpnI restriction sites andunique SailI, BglIII, PstI, and XhoI cloning sites are located betweenthe 5' and 3' noncoding regions.

EXAMPLE 4

Isolation of Other Seed Specific Promoters

Other seed-specific promoters may be isolated from genes encodingproteins involved in seed triacylglycerol synthesis, such as acylcarrier protein from Brassica seeds.

Immature seeds were collected from Brassica campestris cv. "R-500," aself-compatible variety of turnip rape. Whole seeds were collected atstages corresponding approximately to 14 to 28 days after flowering. RNAisolation and preparation of a CDNA bank was as described above for theisolation of a spinach ACP CDNA clone except the vector used waspCGN565. To probe the cDNA bank, the oligonucleotide (5')-ACTTTCTCAACTGTCTCTGGTTTAGCAGC-(3' ) was synthesized using an AppliedBiosystems DNA Synthesizer, model 380A, according to manufacturer'srecommendations. This synthetic DNA molecule will hybridize at lowstringencies to DNA or RNA sequences coding for the amino acid sequence(ala-ala-lys-pro-glu-thr-val-glulys-val). This amino acid sequence hasbeen reported for ACP isolated from seeds of Brassica napus (Slabas etal., 7th International Symposium of the Structure and Function of PlantLipids, University of California, Davis, Calif., 1986); ACP from B.campestris seed is highly homologous. Approximately 2200 different cDNAclones were analyzed using a colony hybridization technique (Taub andThompson, Anal. Biochem. (1982) 126:222-230) and hybridizationconditions corresponding to Wood et al. (Proc. Natl. Acad. Sci. (1985)82:1585-1588). DNA sequence analysis of two cDNA clones showing obvioushybridization to the oligonucleotide probe indicated that one,designated pCGN1Bcs, indeed coded for an ACP-precursor protein by theconsiderable homology of the encoded amino acid sequence with ACPproteins described from Brassica napus (Slabas et al., 1980 supra).Similarly to Example 3, the ACP CDNA clone, pCGN1BCS, was used toisolate ACP genomic clones containing the regulatory information forexpression of ACP during triacylglyceride synthesis in the seeds. DNAwas isolated from B. campestris cv. R500 young leaves by the procedureof Scofield and Crouch (J. Biol.Chem. (1987) 262:12202-12208). A Sau3Apartial genomic library of the B. campestris DNA was made in the lambdavector Embl 3 (Stratagene, San Diego, Calif.) using establishedprotocols (Maniatis et al., (1982) supra) and manufacturer'sinstructions. The titer of the library was -1.0×10⁸ phage/ml. Sixhundred thousand recombinant bacteriophage were plated and screened asdescribed in Example 3 with the exception that the E. coli host cellsused were strain P2392 (Stratagene, San Diego, Calif.). Filters wereprehybridized and hybridized at 42° C. in 25 ml each of hybridizationbuffer containing 50% formamide, 10X Denhardt's, 5X SSC, 5 Mm EDTA, 0.1%SDS, and 100 μg/ml denatured salmon sperm DNA (reagents described inManiatis et al., (1982) supra). The probe used in these hybridizationswas 0.2 μg of a nick-translated 530 base pair BglII-DraI fragment ofpCGNlBcs, the B. campestris ACP cDNA clone described above. Six plaqueswere hybridized strongly on duplicate filters after washing the filtersat 55° C. in 0.1X SSC/0.2 % SDS, and were plaque-purified as described(Maniatis et al., (1982) supra).

Restriction analysis followed by Southern hybridization was performed onsome of the clones using the hybridization conditions and radiolabeledprobe described above. One clone, Bcg4--4, contains the ACP gene on twooverlapping restriction fragments, an ˜5.1 kb SstI fragment and an ˜1.2kb HindIII fragment. These restriction fragments were subcloned into thecloning vector pCGN565. The DNA sequence of some regions of thesubclones verified by homology that Bcg4-4 is an ACP gene. The sequencealso shows that this particular ACP gene is expressed in plants, as thesequence in the coding region matches exactly the sequence of thePCGNLBcs ACP cDNA except for three regions. These regions are believedto be intervening sequences, a common element of eukaryotic genes thatis spliced out during processing of mRNA (Padgett et al., Ann. Rev.Biochem. (1986) 55:1119-1150). Further restriction mapping of the SstIsubclone identified an XhoI fragment containing ˜1.5 kb of 5' sequenceupstream from the XhoI site near the 5' end of the PCGNLBCS CDNA clone.This XhoI fragment was subcloned in opposite orientations in thecloning/sequencing vector Bluescript+(Stratagene, San Diego, Calif.) andthe clones were designated pCGN1941 and pCGN1941' . DNA sequencing of 1kb of the DNA upstream of the coding region was completed. Also, thecomplete sequence of the 1.2 kb HindIII subclone described above wasdetermined. The DNA sequence derived from the clones described above isshown in FIG. 3. Additional sequences at the 3' end of the ACP gene weresubcloned on an ˜1.6 kb SstI-BglII fragment into Bluescript+andBluescript - (clones are designated pCGN1940 and pCGN1940' ). The SstIsite in these clones is the one found at the 3' end of the ACP codingregion of PCGN1Bcs.

An expression cassette can be constructed from the 5' upstream sequencesand 3' downstream sequences of Bcg4-4 as follows. The PCGN1941 XhoIsubclone is used for the 5' regulatory region. This clone contains theXhoI insert in the opposite orientation of the lacZ gene. The 3'regulatory region is altered to allow cloning as a PstIBglII fragmentinto PCGN565 by oligonucleotide site-directed mutagenesis.Single-stranded DNA is made from pCGN1940 and altered by mutagenesis asdescribed (Adelman et al., supra) with the synthetic oligonucleotide 5'CTTAAGAAGTAACCCGGGCTGCAGTTTTAGTATTAAGAG 3' . This oligonucleotideprovides SmaI and PstI restriction sites just after the TAA stop codonof the pCGNlBcs cDNA. The PstI-BglII 3' fragment is then cloned into thePstI and BamHI sites (the BamHI restriction site is destroyed in thisprocess) of PCGN565. The resulting clone is digested with PstI and SmaI,and the fragment inserted into the corresponding sites in PCGN1941(described above) in the same orientation as the 5' region. Theresulting clone comprises the ACP expression cassette with PstI, EcoRI,and EcoRV sites available between the 5' and 3' regulatory regions forthe cloning of genes to be expressed under the regulation of these ACPgene regions.

EXAMPLE 5

Isolation of Seed-specific cDNA Clone. EA9

Ninety-six clones from the 14-28 day postanthesis B. campestris seedcDNA library (described in the previous example) were screened by dotblot hybridization of miniprep DNA on Gene Screen Plus nylon filters(NEN Research Products, Boston, Mass.). The probes used wereradioactively labeled first-strand synthesis cDNAs made from the day14-28 postanthesis seed mRNA or from B. campestris leaf MRNA. Cloneswhich hybridized strongly to seed cDNA and little or not at all to leafcDNA were catalogued. A number of clones were identified as representingthe seed storage protein napin by cross-hybridization with an XhoI-SalIfragment of pNI (Crouch et al., (1983) supra), a B. napus napin cDNA.One of these napin clones, BE5, was used in Example 3 to identify a B.campestris genomic clone as a source of an embryo-specific promoter.

Another abundant class of cDNA clones were those represented by a clonedesignated EA9. EA9 cross-hybridized to seven other cDNA clones of 600cDNAs screened by dot blot hybridization and was highly expressed inseeds and not in leaves. Northern blot analysis of mRNA isolated fromday 14 postanthesis whole seed, and day 21 and 28 postanthesis embryosusing a 700 bp EcoRI fragment of EA9 (see below) as a probe shows thatEA9 is highly expressed at day 14 and expressed at a much lower level atday 21 and day 28 postanthesis. Because the embryo is so small at day14, it was suspected that the predominant expression of EA9 might be ina tissue other than the embryo. Total RNA was isolated (Crouch et al.,(1983) supra) from whole seed (14, 15, 17 and 19 days postanthesis),seed coats (day 14 and day 21 postanthesis) and embryos (day 21postanthesis). Twenty-five μg of each sample were analyzed by Northernblot analysis as described in Example 2. The probe used was a 0.7 kbEcoRI DNA fragment isolated from the EA9 CDNA and labeled bynick-translation. The results of the Northern analysis showed the EA9RNA was detected in whole seed at all times tested and in seed coats,but not in the embryo. A separate Northern analysis of whole seed RNAfrom days 13 through day 31 postanthesis (in two day intervals)indicated that EA9 was highly expressed between days 13 to 21 but wasbarely detectable by day 27 postanthesis.

In Situ Hybridization Seed-coat specific expression of EA9 was confirmedby in situ hybridization analysis. Day 14 and 21 postanthesis wholeseeds of kB. campestris were fixed in a 4% paraformaldehyde phosphatebuffered saline (PBS) solution. The tissue was then dehydrated through agraded tertiarybutyl alcohol (TBA) series, infiltrated with paraplastand cast into paraffin blocks for sectioning (Berlyn and Miksche,Botanical Microtechnique and Cytochemistry (1976), Iowa State UniversityPress). Five μm longitudinal sections of the embedded seeds (onecell-layer thickness) were generated on a Reichert Histostat rotarymicrotome. The paraffin ribbons containing the seed sections were thenaffixed to gelatin-chrome alum subbed slides (Berlyn Miksche, (1976)supra).

Single-stranded radiolabeled RNA probes were made using the Riboprobereaction system (Promega, Madison, Wis.). This system utilizes a vectorwhich is derived from pUC12 and contains a bacteriophage SP6 promoterwhich lies immediately upstream from an M13 polylinker. First, the 700bp EcoRI fragment was isolated from EA9 and subcloned into thepolylinker region of the riboprobe vector in both orientations (senseand anti-sense). To generate a template for the transcription run-offtranscription reactions, the recombinant plasmids were propagated,purified, and linearized with HindIII. The templates were then incubatedin a reaction mixture containing the SP6 RNA polymerase, triphosphatesand ³⁵ S-UTP (as described by the manufacturer). After adding RQ DNase(Promega), the labeled RNAs were run over Boehringer pre-packed Sephadexspin columns to remove unincorporated triphosphates.

The slides containing the sectioned seeds were hybridized with theradiolabeled sense and anti-sense RNA transcripts of EA9 according tothe methods of Singer et al. (Biotechniques (1986) 4:230-241) and Taylorand Martineau (8 Plant. Physiol. (1986) 82:613-618). The hybridizedslides were then treated with nuclear track emulsion NTB-3, (EastmanKodak Company, Kodak Materials for Light Microscope Autoradiography,1986) sealed in a light-tight box and exposed for 4 weeks at 5-10 C.After bringing the slides to room temperature they were developed inD-19 developer (Eastman Kodak Company), rinsed, fixed and dehydratedthrough a graded alcohol series. Cover slips were mounted with cytoseal(VWR Scientific).

Hybridization of the radiolabeled anti-sense EA9 riboprobe was seen onlyin the seed coat tissue of both day 14 and 21 seeds. No hybridization ofthe radiolabeled sense EA9 riboprobe was seen in any seed tissues.

DNA Sequence and Gene Copy Number

The restriction map and sequence of the EA9 CDNA clone have beendetermined (FIG. 4). Identification of a polyadenylation signal(Proudfoot and Brownlee, Nature (1976) 263:211-214) and of polyA tailsat the 3' -end of EA9 indicated the orientation of the cDNA clone andthe direction of transcription of the mRNA. The function of the encodedprotein is unknown at this time.

EA9 s a member of a small gene family as shown by Southern blotanalysis. DNA was isolated from B. campestris leaves (as described inExample I, Southern analysis), digested with either BamHI, BglII orHindIII and probed with a labeled fragment of EA9 . Three fragments ofgenomic DNA hybridized in both BamHI and BglII digests. Only 2 bandshybridized in the HindIII digest. The data suggests that the EA9 familycomprises between one and three genes.

The sequence of EA9 is used to synthesize a probe which identifies aunique class of Brassica seed-specific genes from a genomic library inthe manner described in Examples II and III. The regulatory sequences ofthese genes is used to construct an expression cassette similar to thosedescribed for the napin genes, with the EA9 construct directing seedcoat specific expression of any gene inserted in it.

EXAMPLE 6

Other Seed Specific Examples

Other seed-specific genes also can serve as useful sources of promoters.cDNA clones of cruciferin, the other major seed storage protein of B.napus , have been identified (Simon et. al., (1985) supra) and could beused to screen a genomic library for promoters. Without knowing thespecific functions, yet other cDNA clones can be classified as to theirlevel of expression in seed tissues, their timing of expression (i.e.,when postanthesis they are expressed) and their approximaterepresentation (copy number) in the B. campestris genome. Clones fittingthe criteria necessary for expressing genes related to fatty acidsynthesis or other seed functions can be used to screen a genomiclibrary for genomic clones which contain the 5' and 3' regulatoryregions necessary for expression. The non-coding regulatory regions canbe manipulated to make a tissue-specific expression cassette in thegeneral manner described for other genes in previous examples.

EXAMPLE 7

Construction of Tomato Ripe Fruit cDNA Bank and Screening forFruit-Specific Clones

Tomato plants (Lycopersicon esculentum cv UC82B) were grown undergreenhouse conditions. Poly(A)⁺ RNA was isolated as described by Manssonet al., Mol. Gen. Genet. (1985) 200:356--361. The synthesis of cDNA frompoly(A)^(+RNA) prepared from ripe fruit, cloning into the PstI site ofthe plasmid pUC9 and transformation into an E. coli vector were all asdescribed in Mansson et al., Mol. Gen. Genet. (1985) 200:356-361.

Library Screening

Two thousand recombinant clones were screened by colony hybridizationwith radiolabeled cDNA made from tomato red fruit MRNA, immature greenfruit mRNA, and leaf mRNA. Bacterial colonies immobilized ontoGeneScreen Plus filters (New England Nuclear), were denatured in 1.5 MNaCl in 0.5 M NaOH, then neutralized in 1.5 M NaCl in 0.5 M Tris-HCl pH8, and allowed to air dry. Hybridization, washing and autoradiographywere all performed as described in Maniatis et al., Molecular Cloning: ALaboratory Manual (1982) Cold Spring Harbor, N.Y.

Sixty-five clones were selected which had more intense hybridizationsignals with fruit cDNA than with leaf cDNA and therefore appeared to beunder-represented in the leaf mRNA population relative to the fruitpopulation. Replicate slot blot filters were prepared using purified DNAfrom the selected clones and hybridized with radioactive CDNA from leaf,green fruit, and red fruit as before. This allowed selection of cDNAclone 2A11, also referred to as pCGN1299 which is on at high levels inboth the fruit stages (red and green) and off in the leaf.

EXAMPLE 8 Analysis of Clones

Synthesis of RNA Probes

The CDNA insert of pCGN1299 was excised as an EcoRI to HindIII fragmentof approximately 600 bp (as measured on an agarose gel), and subclonedinto the Riboprobe vector pGEM1 (Promega Biotec), creating pCGN488. ³²P-labeled transcripts made from each strand of the pCGN488 insert usingeither SP6 or T7 polymerase were used as probes in separate Northernblots containing mRNA from leaf, immature green and mature red fruits.The RNA transcript from the SP6 promoter did not hybridize to the tomatomRNA. However, the transcript from the T7 promoter hybridized to an mRNAof approximately 700 nt in length from the green fruit and the red fruitbut not to mRNA from tomato leaf. The direction of transcription of thecorresponding mRNA was thus determined.

The tissue specificity of the pCGN1299 CDNA was demonstrated as follows.RNA from root, stem, leaf, and seven stages of fruit development(immature green, mature green, breaker, turning, pink, light red, andred) was sized on formaldehyde/agarose gels according to the methoddescribed by Maniatis et al., (1982), immobilized on nitrocellulose andhybridized to ³² p-labeled RNA which was synthesized in vitro frompCGN488 using T7 polymerase. Each lane contained 100 ng of polyA⁺ RNAexcept for two lanes (pink and light red lanes) which contained 10 pg oftotal RNA. The Northern analysis of mRNA from toot, stem, leaf, andvarious stages of fruit development indicated that pCGN1299 cDNA wasexpressed in all stages of fruit development from the early stagesimmediately after anthesis to red ripe fruit. No mRNA hybridizing topCGN1299 was found in leaf, stem, or root tissue. The size of the mRNAspecies hybridizing to the pCGN488 probe was approximately 700 nt.

Message abundance corresponding to the pCGN1299 cDNA was determined bycomparing the hybridization intensity of a known amount of RNAsynthesized in vitro from pCGN488 using SP6 polymerase to mRNA from redtomato fruit in a Northern blot. The ³² p-labeled transcript frompCGN488 synthesized in vitro using T7 polymerase was used as a probe.The Northern analysis was compared to standards which indicated that thepCGN1299 cDNA represents an abundant mRNA class in tomato fruit, beingapproximately 1% of the message.

EXAMPLE 9 Sequencing of DCGN1299 and pCGN1298 CDNA Clones

DNA Sequencing

The polyA⁺ sequence was missing from pCGN1299 cDNA. A longer CDNA clone,pCGN1298, therefore was identified by its hybridization with the pCGN488probe. The complete DNA sequence of the two CDNA inserts was determinedusing both Maxam-Gilbert and the Sanger dideoxy techniques and is asfollows. The sequence of pCGN1298 contains additional sequences at boththe 5' and 3' end compared to pCGN1299. As shown in FIG. 8, thesequences are identical over the region that the two clones have incommon.

Amino Acid Sequence

The pCGN1299 cDNA sequence was translated in three frames. The longestopen reading frame (which starts from the first ATG) is indicated. BothpCGN1299 and pCGN1298 have an open reading frame which encodes a 96amino acid polypeptide (see FIG. 8). The protein has a hydrophobicN-terminus which may indicate a leader peptide for protein targeting. Ahydrophobicity profile was calculated using the Hopp and Woods, (Proc.Natl. Acad. Sci. USA (1981) 78:3824-3828) algorithm. Residues 10-23 havean extremely hydrophobic region. A comparison of 2A11 to pea storageproteins and other abundant storage proteins is shown in FIG. 6. Thesulfur-rich composite of the fruit-specific protein is similar to a peastorage protein which has recently been described (see Higgins et al.,J. Biol. Chem. (1986) 261:11124-11130, for references to the individualpeptides). This may indicate a storage role for this fruitspecificprotein abundant species.

EXAMPLE 10 Screening Genomic Library for Genomic Clones

Southern Hybridization

Southern analysis was performed as described by Maniatis et al., 1982.Total tomato DNA from cultivar UC82B was digested with EcoRI or HindIII,separated by agarose gel electrophoresis and transferred tonitrocellulose. Southern hybridization was performed using a ³²p-labeled probe produced by nick translation of pCGN488 (Maniatis etal., 1982). The simple hybridization pattern indicated that the geneencoding pCGN1299 cDNA was present in a few or perhaps even one copy ina tomato genome.

Isolation of a Genomic Clone

A genomic library established in Charon35/Sau3A constructed from DNA ofthe tomato cultivar VFNT-Cherry was screened using the ³² p-!-RNA fromcDNA clone pCGN488 as a probe. A genomic clone containing approximately12.5 kb of sequence from the tomato genome was isolated. The regionwhich hybridizes to a PCGN488 probe spans an XbaI restriction site whichwas found in the cDNA sequence and includes the transcriptionalinitiation region designated 2A11.

Sequence of Genomic Clone

The DNA sequence of the genomic clone was determined by Sanger dideoxytechniques and is as shown in FIG. 7. The sequence of the genomic cloneis identical to the pCGN1299 cDNA clone over the region they have incommon.

Subcloning

The region surrounding the XbaI restriction site, approximately 2.4 kbin the 5' direction and approximately 2.1 kb in the 3' direction wassubcloned to provide an expression cassette. The 5' XhoI to XbaIfragment and the 3' XbaI to EcoRI fragment from the 2A11 genomic clonewere inserted into a pUC-derived chloromphenicol plasmid containing aunique XhoI site and no XbaI site. This promoter cassette plasmid iscalled pCGN1273.

EXAMPLE 11 Construction of Fruit-Specific Antisense Cassette

Insertion of Antisense Fragment

The 2A11 genomic fragment was tagged with PG antisense sequences byinsertion of PG into the unique XbaI site of the pCGN1273 promotercassette in the antisense orientation. The inserted sequences increasedthe size of the mRNA over the endogenous transcript, and thus theexpression pattern of the construct could be compared to the endogenousgene by a single Northern hybridization in a manner analogous to thedetection of a tuber-specific potato gene described by Eckes et al.,Mol. Gen. Genet. 1986 205:14-22.

EXAMPLE 12 Insertion of Tagged Genomic Construction Into AgrobacteriumBinary Vectors

The tagged genomic construction is excised using the flanking XhoIrestriction enzyme sites and is cloned into the unique SalI site of thebinary plasmid pCGN783 (see Example 2 for construction) containing aplant kanamycin resistance marker between the left and right borders toprovide plasmid pCGN1269.

This plasmid binary vector in E. coli C2110 is conjugated into A.tumefaciens containing a disarmed Tiplasmid capable of transferring thepolygalacturonase antisense cassette and the kanamycin resistancecassette into the plant host genome.

The Agrobacterium system which is employed is A. tumefaciens PC2760 (G.Ooms et al., Plasmid (1982) 7:15-29; Hoekema et al., Nature (1983)303:179-181; European Patent Application 84-200239.6, 2424183).

EXAMPLE 13 Transfer of Genomic Construction to Tomato via Cocultivation

Substantially sterile tomato cotyledon tissue is obtained from seedlingswhich have been grown at 24° C., with a 16 hr/8 hr day/night cycle in100×25 mm petri dishes containing Murashige-Skoog salt medium and 0.8%agar (pH 6.0). Any tomato species may be used, however, here the inbredbreeding line was UC82B, available from the Department of VegetableCrops, University of California, Davis, Calif. 95616. The cotyledons arecut into three sections and the middle placed onto feeder plates for a24-hour preincubation. The feeder plates are prepared by pipetting 0.5ml of a tobacco suspension culture (10⁶ cells/ml) onto 0.8% agar medium,containing Murashige minimal organic medium (K.C. Biologicals), 2,4-D(0.1 mg/1), kinetin (1 mg/1), thiamine (0.9 mg./1) and potassium acidphosphate (200 mg/I, pH 5.5). The feeder plates are prepared two daysprior t use. A sterile 3 mm filter paper disk containing feeder mediumis placed on top of the tobacco cells after the suspension cells aregrown for two days.

Following the preincubation period, the middle one third of thecotyledon sections are placed into a liquid MG/L broth culture (1-5 ml)of the A. tumefaciens strain. The binary plasmid pCGN1269 is transferredto A. tumefaciens strain 2760 by conjugation or by transformationselecting for Gentamicin resistance encoded by the plasmid pCGN1269. Thecotyledon sections are cocultivated with the bacteria for 48 hrs. on thefeeder plates and then transferred to regeneration medium containing 500mg/l carbenicillin and 100 mg/1 kanamycin. The regeneration medium is aK.C. Biologicals Murashige-Skoog salts medium with zeatin (2 mg/1)myo-inositol (100 mg/1), sucrose (20 g/1), Nitsch vitamins andcontaining 0/8% agar (pH 6.0). In 2-3 weeks, shoots are observed todevelop. When the shoots are approximately 1.25 cm, they are excised andtransferred to a Murashige and Skoog medium containing carbenicillin(500 mg/1) and kanamycin (50 mg/1) for rooting. Roots develop within10-12 days.

Shoots which develop and subsequently root on media containing thekanamycin are tested for APH3' II enzyme.

An aminoglycoside phosphotransferase enzyme (APH3' II) assay isconducted on putative transformed tomato plants and shoots. APH3' IIconfers resistance to kanamycin and neomycin. APH3' II' s activity isassayed (Reiss et al., Gene (1984) 30:211-218) employing electrophoreticseparation of the enzyme from other interfering proteins and detectionof its enzymatic activity by in situ phosphorylation of kanamycin. Bothkanamycin and γ⁻³² P! ATP act as substrates and are embedded in anagarose gel which is placed on top of the polyacrylamide gel containingthe proteins. After the enzymatic reaction, the phosphorylated kanamycinis transferred to P-81 phosphocellulose ion exchange paper and theradiolabeled kanamycin is finally visualized by autoradiography. TheReiss et al., method is modified in the final washing of the P-81 ionexchange paper by rinsing in 0.1 mg/ml of proteinase K.

EXAMPLE 14 Construction of Taaged 2A11 Plasmids In Binary Vectors

The compete sequence of the 2A11 genomic DNA cloned into pCGN1273 fromthe XhoI site (position 1 at the 5' end) to the EcoRI site (position4654) is shown in FIG. 7. pCGN1267 was constructed by deleting frompCGN1273 a portion of the plasmid polylinker from the EcoRV site to theBamHI site. Two DNA sequences were inserted into pCGN1273 at the uniqueXbaI site (position 2494). This site is in the 3' non-coding region ofthe 2A11 genomic clone before the poly A site. pCGN1273 was tagged with360 bp (from base number 1 to 360) from the 5' region of the tomatopolygalacturonase (PG) cDNA clone, Fl (Sheehy et al., Mol. Gen. Genet.(1987) 208:30-36) at the unique XbaI restriction enzyme site. The tagwas inserted in the antisense orientation resulting in plasmid pCGN1271and in the sense orientation yielding plasmid pCGN1270. Each plasmid waslinearized at the unique BglII restriction enzyme site and cloned intothe binary vector pCGN783 at the unique BamHI restriction enzyme site.pCGN1273 was also tagged with a 0.5 kb fragment of DNA (base number 1626to 2115) from a PG genomic clone (see FIG. 8) which spans the 5' end ofintron/exon junction. This fragment was cloned into the XbaI siteresulting in plasmid pCGN1215. pCGN1215 was linearized at the uniqueBglII site and cloned into pCGN783 at the BamHI site resulting in twoplasmids, pCGN1219 and pCGN1220, which differ only in the orientation ofpCGN1215 within pCGN783.

Three DNA sequences were inserted into pCGN1267 at the uniquerestriction enzyme sites (position 2402, 2406). These sites are in the3' non-coding region of the 2A11 genomic clone, 21 bp from the stopcodon. The 383 bp XbaI fragment from the PG cDNA clone was cloned intothe ClaI site of pCGN1267 after filling in the XbaI and ClaI ends withKlenow and blunt ligation. The fragment in a sense orientation resultedin plasmid pCGN1263 and in the antisense orientation gave pCGN1262.pCGN1263 was linearized at the unique BglII site and cloned into pCGN783at the BamHI site yielding pCGN1260. pCGN1262 was also linearized at theBglII site and cloned into pCGN783 at the BamHI site resulting in twoplasmids, pCGN1255 and pCGN1258, which differ only in the orientation ofpCGN1262 in the binary vector pCGN783.

The 0.5 kb fragment of the PG genomic clone spanning the intron/exonjunction (supra) was cloned into pCGN1267 at the ClaI site in anantisense direction yielding plasmid pCGN1225. This plasmid waslinearized at the BglII restriction enzyme site and cloned in to pCGN783at the BamHI site producing two plasmids, pCGN1227 and pCGN1228, whichdiffer only in the orientation of pCGN1225 in the binary vector.

The Eco7 fragment (base numbers 5545 to 12,823) (Barker et al., PlantMol. Biol. (1983) 2:335-350) from the octopine plasmid pTiA6 of A.tumefaciens (Knauf and Nester, Plasmid (1982) 8:45-54) was subclonedinto pUC19 at the EcoRI site resulting in plasmid pCGN71. A RsaI digestallowed a fragment of DNA from bases 8487 to 9036 of the Eco7 fragmentto be subcloned into the vector m13 BlueScript Minus (Stratagene, Inc.)at the SmaI site resulting in plasmid pCGN1278. This fragment containsthe coding region of the genetic locus designated tmr which encodes adimethylallyl transferase (isopentenyl transferase) (Akiyoshi et al.,Proc. Natl. Acad. Sci. USA (1984) 81:5994-5998; Barry et al., ibid(1984) 81:4776-4780). An exonuclease/mung bean treatment (PromegaBiotech) produced a deletion on the 5' end of the tmr gene to a point 39base pairs 5' of the start codon. The tir gene from pCGN1272 wassubcloned into the ClaI site of pCGN1267. The tmr gene in the senseorientation yielded pCGN1261 and in the antisense orientation gaveplasmid pCGN1266. pCGN1261 was linearized at the BglII site and clonedinto pCGN783 at the BamHI site resulting in plasmid pCGN1254. pCGN1266was also linearized at the BglII site and subcloned into pCGN783 at theBamHI site yielding two plasmids, pCGN1264 and pCGN1265, which differonly in the orientation of pCGN1266 in pCGN783.

Analysis of Expression in Transgenic Plants

Immature green fruit (approximately 3.2 cm in length) was harvested fromtwo tomato plants cv. UC82B that had been transformed with a disarmedAgrobacterium strain containing pCGN1264. Transgenic plants aredesignated 12641 and 1264-11. The pericarp from two fruits of each plantwas ground to a powder under liquid N₂, total RNA extracted and polyA⁺mRNA isolated (as described in Mansson et al., Mol. Gen. Genet. (1985)200:356-361). Young green leaves were also harvested from each plant andpolyA⁺ mRNA isolated.

Approximately 19 μg of total RNA from fruit, 70 ng of polyA⁺ MRNA fromfruit and 70 ng of polyA⁺ mRNA from leaves from transformed plants1264-1 and 1264-11 was run on a 0.7% agarose formaldehyde Northern geland blotted onto nitrocellulose (Maniatis et al., Molecular Cloning: ALaboratory Manual (1982) Cold Spring Harbor, N. Y.). Also included onthe gel as a negative control was approximately 50 ng of polyA⁺ mRNAfrom leaf and immature green fruit of a nontransformed UC82B plant.

As a positive control and to help in quantitating mRNA levels, in vitrotranscribed RNA from pCGN1272 was synthesized using T3 polymerase(Stratagene, Inc.). Nineteen pg and 1.9 pg of this in vitro synthesizedRNA were loaded on the Northern gel.

The probe for the Northern filter was the 1.0 kb tmr insert DNA (a KpnIto SACI fragment) from pCGN1272 isolated by electroelution from anagarose gel (Maniatis, supra (1982)) and labeled by nick translation(Bethesda Research Laboratory kit) using α³² p dCTP (Amersham).

The Northern filter was prehybridized at 42° C. for 5 hrs. in thefollowing solution: 25 ml formamide, 12.5 ml 20X SSC, 2.5 ml 1 M NaP, 5ml 50X Denhardts, 0.5 ml 10% SDS, 1 ml 250 mM EDTA, 1 ml 10 mg/ml ssDNAand 2 ml H₂ O. Then onefifth volume of 50% dextran sulfate andapproximately 2.2X 107 cpm of the probe was added and hybridization wasfor 15 hrs. at 42° C.

The Northern filter was washed one time in 2X SSC and 0.1% SDS at 550for 20 minutes each wash. The filter was allowed to air dry before beingplaced with Kodak XAR film and an intensifying screen at -700 for twodays.

Northern Results on Transgenic Plants The nicked tmr probe hybridizedwith a mRNA species approximately 1.7 kb in length was observed in thetotal RNA and polyA⁺ mRNA fruit lanes of the Northern blot. This is theexpected length of the reintroduced 2A11 gene (0.7 kb) tagged with thetmr gene (1.0 kb) in the antisense orientation. The level of expressionfrom the reintroduced tagged gene is somewhat lower than the level ofexpression of the endogenous 2A11 gene. The level of expression of thereintroduced gene in immature green fruit is higher than the expressionlevel in leaf tissue with a small amount of hybridizing mRNA in leaftissue in these transformants.

EXAMPLE 15 Different Sized 2A11 5' Regions

The design of the 2A11 cassette is shown in FIG. 9. The cassettecontains 3.8 kb of DNA 5' of the transcriptional start site and theentire 3' region (from the TGA stop codon to a site 2.0 kb 3' of thepoly A addition site) of the 2A11 gene. FIG. 7 shows the restrictionsites and indicates (below the representation of the gene) the regionsof the 2A11 gene used to construct the 2A11 cassette. The 2A11 cassettewas constructed as follows.

Transcriptional Initiation Region

The 5' end of the 2A11 cassette was constructed starting with an EcoRIsubclone genomic clone as described in application PCTUS88/01811 clonedinto the EcoRI of Bluescript (+) (Stratagene) resulting in pCGN1288.This clone contains sequences from the EcoRI site at position 1651 inthe intron of the 2A11 gene to the EcoRI site located 2.5 Kb upstream ofthe XhoI site at position 1 of the sequenced region (see FIG. 7). TheXHOI fragment from position 1 of the sequenced region to the XHOI sitein the Bluescript polylinker was deleted creating plasmid pCGN2004 whichcontain the 2A11 region from position 1 to position 1651. The codingregion of 2A11 was deleted by treating this plasmid withExonucleaseIII/S1 using the commercially available Erase-a-Base Kit(Promega Biotec) and sequencing deletion plasmids until one was foundwhich had the coding region deleted to position 1366. The resultingplasmid, pCGN1251, had the genomic region from the XhoI site(position 1) to position 1366. The EcoRI fragment of pCGN1288 was thentransferred to a chloramphenicol resistant plasmid vector, pCGN2015, tomake pCGN1231. pCGN2015 is a Cm resistant derivative of the Bluescriptplasmid. A BstEII/BamHI fragment of pCGN1251 was then transferred intoBstEII/BamHI digested pCGN1231 to make pCGN1235 which contains theregion from the EcoRI site (2.5 kb upstream of the sequenced region) toposition 1366 of the sequenced region flanked by the Bluescriptpolylinker in a Cm resistant vector.

Transcriptional and Translational Termination Region

The 3' end of the 2A11 cassette was constructed from pCGN1273 (describedin application PCT/US8801811) by digesting the plasmid with PvuI andEcoRI, isolating the 2249 bp insert (from position 2402 to 4653),ligating with a double-stranded oligonucleotide containing the sequenceshown in FIG. 7 from the BamHI sticky end to a PvuI sticky end into aBluescript vector which had been digested with BamHI and EcoRI. Theresulting plasmid, pCGN1238, contains the 3' end of the 2A11 gene fromthe stop codon at position 2381 to the EcoRI site at position 4653.

Final Construction

Several versions of the 2A11 cassette in different vectors withdifferent flanking restriction sites have been constructed; maps of theplasmids are shown in FIG. 10.

A cassette containing the 5' and 3' regions of the 2A11 gene wasconstructed by ligating the BamHI to EcoRI insert of pCGN1238 intopCGN1235 which had been digested with BamHI and XbaI (the XbaI sitehaving been filled in with Klenow polymerase to make a blunt-endedfragment). The resulting plasmid, pCGN1240, has the 5' end of the 2A11gene from the EcoRI site 2.5 kb upstream of the XhoI site (position 1)to position 1366 (which is located between the transcriptionalinitiation site of the 2A11 gene and the ATG), followed by a polylinkerregion with sites for SmaI, BamHI, PstI and SalI which can beconveniently used to insert genes followed by the 3' region fromposition 2381 to 4653. The plasmid backbone of pCGN1240 is theBluescript Cm plasmid described above.

Construction of Plasmid pCGN1241

A more convenient version has the EcoRI of pCGN1240 excised and insertedinto a Bluescript vector called pCGN1239 which has an altered polylinkerregion such that the entire cassette can be excised as a SacI-KpnIfragment. The altered Bluescript vector, pCGN1239, was constructed bymodifying the BlueScript polybinder from the SacI site to the KpnI siteincluding a synthetic polylinker with the following sequence:AGCTCGGTACCGAATTCGAGCTCGGTAC to create a polylinker with the followingsites: SacI-KpnI-EcoRI SacIKpnI. The EcoRI insert of pCGN1240 wasinserted into pCGN1239 to make pCGN1241 (see FIG. 9).

Construction of DCGN2610 and pCGN2611

A chloramphenicol resistant version of the 2A11 promoter cassette wasconstructed by inserting the synthetic polylinker described above (seeconstruction of pCGN1241) into pCGN2015 to make pCGN1246, followed byinsertion of the EcoRI fragment of pCGN1241 to make pCGN2610 andpCGN2611 which differ only by the orientation of the inserted fragmentin the plasmid vector (see FIG. 8).

EXAMPLE 16 Comparison of Expression from Different Sized 2A11 5' Regions

A beta-glucuronidase (Gus) reporter gene was used to evaluate the levelof expression and tissue specificity of various 2A11 -Gus constructions.Gus is a useful reporter gene in plant systems because it produces ahighly stable enzyme, there is little or no background (endogenous)enzyme activity in plant tissues, and the enzyme is easily assayed usingfluorescent or spectrophotometric substrates. See, for example,Jefferson Plant Mol. Biol. Rep. (1988) 5:387-405. Histochemical stainsfor Gus enzyme activity are also available which can be used to analyzethe pattern of enzyme expression in transgenic plants. Jefferson (1988),supra.

Constructions containing 1.3 kb (short), 1.8 kb (intermediate length),or 3.8 kb (long) 2A11 5' sequences fused to the Gus reporter gene wereprepared. In addition, constructions were prepared which have altered 3'ends. The altered 3' ends are either a shorter 2A11 3' end from tr5 ofthe T-DNA of the Ti plasmid (Willmitzer et al., Embo. J. (1982)1:139-146; Willmitzer et al., Cell (1983) 42:1045-1056. Theconstructions were transferred to a binary vector (pCGN1578), and usedin A. Tumefaciens cocultivations. The resulting binary was used totransform tomato plants. The transgenic plants obtained werefluorometrically analyzed for Gus enzyme activity.

EXAMPLE 17 Screening Genomic Library for Polygalacturonase GenomicClones

Isolation of a Genomic Clone

An EcoRI partial genomic library established in Charon 4 constructedfrom DNA of a Lycopersicon esculentum cultivar was screened using aprobe from the polygalacturonase CDNA (Sheehy et al., Mol. Gen. Genet.(1987) 208:30-36). A lambda clone containing an approximately 16 kbinsert was isolated from the library, of which an internal 2207 bpHindIIl to EcoRI was sequenced. The HindIII-EcoRI fragment includes thepolygalacturonse promoter region.

Sequence of Genomic Clone

The DNA sequence of the genomic clone was determined by Sanger dideoxytechniques and is as shown in FIG. 8. The sequence of the genomic clonebases 1427 to 1748 are homologous to the polygalacturonase cDNAsequence.

The above results demonstrate the ability to identify inducibleregulatory sequences in a plant genome, isolate the sequences andmanipulate them. In this way, the production of transcription cassettesand expression cassettes can be produced which allow for differentiatedcell production of the desired product. Thus, the phenotype of aparticular plant part may be modified, without requiring that theregulated product be produced in all tissues, which may result invarious adverse effects on the growth, health, and productioncapabilities of the plant. Particularly, fruit-specific transcriptioninitiation capability is provided for modifying the phenotypicproperties of a variety of fruits to enhance properties of interest suchas processing, organoleptin properties, storage, yield, or the like.Further, the results demonstrate one can use transcriptional initiationregions associated with the transcription of sequences in seeds inconjunction with sequences other than the normal sequence to produceendogenous or exogenous proteins or modulate the transcription ofexpression of nucleic acid sequences. In this manner, seeds can be usedto produce novel products, to provide for improved protein compositions,to modify the distribution of fatty acid, and the like.

It is also evident from the above results that not only can soybean betransformed, so as to introduce heterologous genes, but transformedsoybean cells may be regenerated into plants and the plants demonstratethe phenotype of the heterologous gene. In addition, native promoterscan find use in conjunction with heterologous genes and retain theircapability to be induced in the same manner as the native gene.Therefore, one can provide for regulated expression of a heterologousgene, where regulation may be by an external condition, such as light.Furthermore, Ti- or Ri-DNA may be employed for introducing theheterologous gene as part of an expression cassette into the soybeancell without formation of a tumor and the resulting cells grown inculture and plants regenerated from the cells. By appropriate choice ofvarious genes, various properties of the cell may be enhanced byintroduction of additional copies of a homologous gene or new phenotypesmay be provided by expression of heterologous genes. In addition,mutated genes may be employed which can impart novel properties to thehost cell, providing for host resistance to biocides, enhancedproduction of specific metabolites or products at the same or differenttimes from the normal regulated expressions, or the like.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for obtaining a plant having a modifiedphenotype, said method comprising;transforming a host plant cell with aDNA construct under genomic integration conditions, wherein saidconstruct comprises as operably linked components in the direction oftranscription, a promoter region obtainable from a gene, whereintranscription of said gene is light-inducible in a plant chloroplastcontaining tissue, a DNA sequence of interest other than the nativecoding sequence of said gene, and a transcription termination region,wherein said components are functional in a plant cell, whereby said DNAconstruct becomes integrated into a genome of said plant cell;regenerating a plant from said transformed plant cell, and growing saidplant under conditions whereby said DNA sequence of interest isexpressed and a plant having said modified phenotype is obtained.
 2. Amethod for altering the phenotype of chloroplast containing plant tissueas distinct from other plant tissue, said method comprising:growing aplant, wherein said plant comprises cells containing a DNA constructintegrated into their genome, said DNA construct comprising, in the 5'to 3' direction of transcription, a transcriptional initiation regionfrom a gene, wherein transcription of said gene is light-inducible in aplant chloroplast containing tissue, a DNA sequence of interest otherthan the coding sequence native to said transcriptional initiationregion, and a transcriptional termination region, whereby said DNAsequence of interest is transcribed under transcriptional control ofsaid transcriptional initiation region and a plant having an alteredphenotype is obtained.
 3. The method according to claim 1 or 2, whereinsaid DNA construct is flanked by T-DNA.
 4. The method according to claim3, wherein said plant is a soybean, tomato, or rapeseed plant.
 5. Themethod of claim 1 or 2, wherein said gene is an SSU gene.
 6. The methodaccording to claim 1 or 2 or wherein said DNA sequence of interestencodes an enzyme.
 7. The method according to claim 1 or 2 wherein saidDNA sequence of interest is an antisense sequence.
 8. The methodaccording to claim 2, wherein said transcriptional initiation regionfurther comprises a translational initiation region and said DNAsequence of interest is an open reading frame encoding an amino acidsequence.